Antibodies against human angiopoietin 2

ABSTRACT

The present invention relates to a method of treating a disease or disorder in a patient comprising administering, to a patient in need of such treatment, an antibody which binds specifically to human angiopoietin-2 (ANG-2), wherein said antibody comprises:
         (A) a heavy chain variable domain which comprises a CDR3 region of SEQ ID NO: 1, a CDR2 region of SEQ ID NO: 2 and a CDR1 region of SEQ ID NO: 3;   (B) a light chain variable domain which comprises a CDR3 region of SEQ ID NO: 4, a CDR2 region of SEQ ID NO: 5 and a CDR1 region of SEQ ID NO: 6.       

     The present invention also relates to a method for preventing metastasis in a patient suffering from primary cancer comprising administering, to a patient in need of such preventative treatment, an antibody which binds specifically to human angiopoietin-2 (ANG-2), wherein said antibody comprises:
             (A) a heavy chain variable domain which comprises a CDR3 region of SEQ ID NO: 1, a CDR2 region of SEQ ID NO: 2 and a CDR1 region of SEQ ID NO: 3;   (B) a light chain variable domain which comprises a CDR3 region of SEQ ID NO: 4, a CDR2 region of SEQ ID NO: 5 and a CDR1 region of SEQ ID NO: 6.

PRIORITY TO RELATED APPLICATION(S)

This application is a continuation application of U.S. application Ser.No. 13/358,813, which is a divisional application of U.S. applicationSer. No. 12/635,825, filed Dec. 11, 2009, now U.S. Pat. No. 8,133,979,which claims the benefit of European Patent Application No. 08021835.7,filed Dec. 16, 2008. The entire contents of the above-identifiedapplications are hereby incorporated by reference.

BACKGROUND OF THE INVENTION

Angiogenesis is implicated in the pathogenesis of a variety of disorderswhich include solid tumors, intraocular neovascular syndromes such asproliferative retinopathies or age-related macular degeneration (AMD),rheumatoid arthritis, and psoriasis (Folkman, J., et al., J. Biol. Chem.267 (1992) 10931-10934; Klagsbrun, M., et al., Annu Rev. Physiol. 53(1991) 217-239; and Garner, A., Vascular diseases, In: Pathobiology ofocular disease, A dynamic approach, Garner, A., and Klintworth, G. K.(eds.), 2nd edition, Marcel Dekker, New York (1994), pp 1625-1710). Inthe case of solid tumors, the neovascularization allows the tumor cellsto acquire a growth advantage and proliferative autonomy compared to thenormal cells. Accordingly, a correlation has been observed betweendensity of microvessels in tumor sections and patient survival in breastcancer as well as in several other tumors (Weidner, N., et al., N. Engl.J. Med. 324 (1991) 1-8; Horak, E. R., et al., Lancet 340 (1992)1120-1124; and Macchiarini, P., et al., Lancet 340 (1992) 145-146).

ANG-2 and Anti-ANG-2 Antibodies

Human angiopoietin-2 (ANG-2) (alternatively abbreviated with ANGPT2 orANG2) (SEQ ID No: 107) is described in Maisonpierre, P. C., et al.,Science 277 (1997) 55-60 and Cheung, A. H., et al, Genomics 48 (1998)389-91. The angiopoietins-1 and -2 (ANG-1 (SEQ ID No: 108) and ANG-2(SEQ ID No: 107) were discovered as ligands for the Ties, a family oftyrosine kinases that is selectively expressed within the vascularendothelium. Yancopoulos, G. D., et al., Nature 407 (2000) 242-48. Thereare now four definitive members of the angiopoietin family.

Angiopoietin-3 and -4 (Ang-3 and Ang-4) may represent widely divergedcounterparts of the same gene locus in mouse and man. Kim, I., et al.,FEBS Let, 443 (1999) 353-56; Kim, I., et al., J Biol Chem 274 (1999),26523-28. ANG-1 and ANG-2 were originally identified in tissue cultureexperiments as agonist and antagonist, respectively (see for ANG-1:Davies, S., et al., Cell, 87 (1996) 1161-1169; and for ANG-2:Maisonpierre, P. C., et al., Science 277 (1997) 55-60). All of the knownangiopoietins bind primarily to Tie2, and both Ang-1 and -2 bind to Tie2with an affinity of 3 nM (Kd). Maisonpierre, P. C., et al., Science 277(1997) 55-60. Ang-1 was shown to support EC survival and to promoteendothelium integrity, Davis, S., et al., Cell, 87 (1996) 1161-1169;

Kwak, H. J., et al., FEBS Lett 448 (1999) 249-53; Suri, C., et al.,Science 282 (1998) 468-71; Thurston, G., et al., Science 286 (1999)2511-14; Thurston, G., et al., Nat. Med. 6 (2000) 460-63, whereas ANG-2had the opposite effect and promoted blood vessel destabilization andregression in the absence of the survival factors VEGF or basicfibroblast growth factor. Maisonpierre, P. C., et al., Science 277(1997) 55-60. However, many studies of ANG-2 function have suggested amore complex situation. ANG-2 might be a complex regulator of vascularremodeling that plays a role in both vessel sprouting and vesselregression. Supporting such roles for ANG-2, expression analyses revealthat ANG-2 is rapidly induced, together with VEGF, in adult settings ofangiogenic sprouting, whereas ANG-2 is induced in the absence of VEGF insettings of vascular regression. Holash, J., et al., Science 284 (1999)1994-98; Holash, J., et al., Oncogene 18 (1999) 5356-62. Consistent witha context-dependent role, ANG-2 specifically binds to the sameendothelial-specific receptor, Tie-2, which is activated by Ang-1, buthas context-dependent effects on its activation. Maisonpierre, P. C., etal., Science 277 (1997) 55-60.

Corneal angiogenesis assays have shown that both ANG-1 and ANG-2 hadsimilar effects, acting synergistically with VEGF to promote growth ofnew blood vessels. Asahara, T., et al., Circ. Res., 83, (1998) 233-40.The possibility that there was a dose-dependent endothelial response wasraised by the observation that in vitro at high concentration, ANG-2 canalso be pro-angiogenic. Kim, I., et al., Oncogene 19 (2000) 4549-52. Athigh concentration, ANG-2 acts as an apoptosis survival factor forendothelial cells during serum deprivation apoptosis through activationof Tie2 via PI-3 Kinase and Akt pathway. Kim, I., et al., Oncogene 19(2000) 4549-52.

Other in vitro experiments suggested that during sustained exposure, theeffects of ANG-2 may progressively shift from that of an antagonist toan agonist of Tie2, and at later time points, it may contribute directlyto vascular tube formation and neovessel stabilization.Teichert-Kuliszewska, K., et al., Cardiovasc. Res. 49 (2001) 659-70.Furthermore, if ECs were cultivated on fibrin gel, activation of Tie2with ANG-2 was also observed, perhaps suggesting that the action ofANG-2 could depend on EC differentiation state. Teichert-Kuliszewska;K., et al., Cardiovasc. Res. 49 (2001) 659-70. In microvascular ECcultured in a three-dimensional collagen gel, ANG-2 can also induce Tie2activation and promote formation of capillary-like structures.Mochizuki, Y., et al., J. Cell. Sci. 115 (2002) 175-83. Use of a 3-Dspheroidal coculture as an in-vitro model of vessel maturationdemonstrated that direct contact between ECs and mesenchymal cellsabrogates responsiveness to VEGF, whereas the presence of VEGF and ANG-2induced sprouting. Korff, T., et al., Faseb J. 15 (2001) 447-57. Etoh,T., et al. demonstrated that ECs that constitutively express Tie2, theexpression of MMP-1, -9 and u-PA were strongly upregulated by ANG-2 inthe presence of VEGF. Etoh, T., et al., Cancer Res. 61 (2001) 2145-53.With an in vivo pupillary membrane model, Lobov, I. B., et al. showedthat ANG-2 in the presence of endogenous VEGF promotes a rapid increasein capillary diameter, remodeling of the basal lamina, proliferation andmigration of endothelial cells, and stimulates sprouting of new bloodvessels. Lobov, I. B., et al., Proc. Natl. Acad. Sci. USA 99 (2002)11205-10. By contrast, ANG-2 promotes endothelial cell death and vesselregression without endogenous VEGF. Lobov, I. B., et al., Proc. Natl.Acad. Sci. USA 99 (2002) 11205-10. Similarly, with an in vivo tumormodel, Vajkoczy, P., et al. demonstrated that multicellular aggregatesinitiate vascular growth by angiogenic sprouting via the simultaneousexpression of VEGFR-2 and ANG-2 by host and tumor endothelium. Vajkoczy,P., et al., J. Clin. Invest. 109 (2002) 777-85. This model illustratedthat the established microvasculature of growing tumors is characterizedby a continuous remodeling, putatively mediated by the expression ofVEGF and ANG-2. Vajkoczy, M. A., et al., J. Clin. Invest. 09 (2002)777-85.

Knock-out mouse studies of Tie-2 and Angiopoietin-1 show similarphenotypes and suggest that Angiopoietin-1 stimulated Tie-2phosphorylation mediates remodeling and stabilization of developingvessel, promoting blood vessel maturation during angiogenesis andmaintenance of endothelial cell-support cell adhesion (Dumont, D. J., etal., Genes & Development, 8 (1994) 1897-1909; Sato, T. N., Nature, 376(1995) 70-74; (Thurston, G., et al., Nature Medicine 6 (2000) 460-463).The role of Angiopoietin-1 is thought to be conserved in the adult,where it is expressed widely and constitutively (Hanahan, D., Science,277 (1997) 48-50; Zagzag, D., et al., Exp Neurology, 159 (1999)391-400). In contrast, Angiopoietin-2 expression is primarily limited tosites of vascular remodeling where it is thought to block theconstitutive stabilizing or maturing function of Angiopoietin-1,allowing vessels to revert to, and remain in, a plastic state which maybe more responsive to sprouting signals (Hanahan, D., 1997; Holash, J.,et al., Orzcogerze 18 (1999) 5356-62; Maisonpierre, P. C., 1997).Studies of Angiopoietin-2 expression in pathological angiogenesis havefound many tumor types to show vascular Angiopoietin-2 expression(Maisonpierre, P. C., et al., Science 277 (1997) 55-60). Functionalstudies suggest Angiopoietin-2 is involved in tumor angiogenesis andassociate Angiopoietin-2 overexpression with increased tumor growth in amouse xenograft model (Ahmad, S. A., et al., Cancer Res., 61(2001)1255-1259). Other studies have associated Angiopoietin-2overexpression with tumor hypervascularity (Etoh, T., et al., CancerRes. 61 (2001) 2145-53; Tanaka, F., et al., Cancer Res. 62 (2002)7124-29).

In recent years Angiopoietin-1, Angiopoietin-2 and/or Tie-2 have beenproposed as possible anti-cancer therapeutic targets. For example U.S.Pat. Nos. 6,166,185, 5,650,490 and 5,814,464 each disclose anti-Tie-2ligand and receptor antibodies. Studies using soluble Tie-2 werereported to decrease the number and size of tumors in rodents (Lin, P,1997; Lin, P., 1998). Siemester, G., et al. (1999) generated humanmelanoma cell lines expressing the extracellular domain of Tie-2,injected these into nude mice and reported soluble Tie-2 to result insignificant inhibition of tumor growth and tumor angiogenesis. Givenboth Angiopoietin-1 and Angiopoietin-2 bind to Tie-2, it is unclear fromthese studies whether Angiopoietin-1, Angiopoietin-2 or Tie-2 would bean attractive target for anti-cancer therapy. However, effectiveanti-Angiopoietin-2 therapy is thought to be of benefit in treatingdiseases such as cancer, in which progression is dependant on aberrantangiogenesis where blocking the process can lead to prevention ofdisease advancement (Folkman, J., Nature Medicine. 1, (1995) 27-31.

In addition some groups have reported the use of antibodies and peptidesthat bind to Angiopoietin-2. See, for example, U.S. Pat. No. 6,166,185and US 2003/10124129. WO 03/030833, WO 2006/068953, WO 03/057134 or US2006/0122370.

Study of the effect of focal expression of Angiopoietin-2 has shown thatantagonizing the Angiopoietin-1/Tie-2 signal loosens the tight vascularstructure thereby exposing ECs to activating signals from angiogenesisinducers, e.g. VEGF (Hanahan, 1997). This pro-angiogenic effectresulting from inhibition of Angiopoietin-1 indicates thatanti-Angiopoietin-1 therapy would not be an effective anti-cancertreatment.

ANG-2 is expressed during development at sites where blood vesselremodeling is occurring. Maisonpierre, P. C., et al., Science 277 (1997)55-60. In adult individuals, ANG-2 expression is restricted to sites ofvascular remodeling as well as in highly vascularized tumors, includingglioma, Osada, H., et al., Int. J. Oncol. 18 (2001) 305-09; Koga, K., etal., Cancer Res. 61 (2001) 6248-54, hepatocellular carcinoma, Tanaka,S., et al, J. Clin. Invest. 103 (1999) 341-45, gastric carcinoma, Etoh,T., et al., Cancer Res. 61 (2001) 2145-53; Lee, J. H., et al, Int. J.Oncol. 18 (2001) 355-61, thyroid tumor, Bunone, G., et al., Am J Pathol155 (1999) 1967-76, non-small cell lung cancer, Wong, M. P., et al.,Lung Cancer 29 (2000) 11-22, and cancer of colon, Ahmad, S. A., et al.,Cancer 92 (2001) 1138-43, and prostate Wurmbach, J. H., et al.,Anticancer Res. 20 (2000) 5217-20. Some tumor cells are found to expressANG-2. For example, Tanaka, S., et al., J. Clin. Invest. 103 (1999)341-45 detected ANG-2 mRNA in 10 out of 12 specimens of humanhepatocellular carcinoma (HCC). Ellis' group reported that ANG-2 isexpressed ubiquitously in tumor epithelium. Ahmad, S. A., et al., Cancer92 (2001) 1138-43. Other investigators reported similar findings. Chen,L., et al., J. Tongji Med. Univ. 21 (2001) 228-30, 235 (2001). Bydetecting ANG-2 mRNA levels in archived human breast cancer specimens,Sfilogoi, C., et al., Int. J. Cancer 103 (2003) 466-74 reported thatANG-2 mRNA is significantly associated with auxiliary lymph nodeinvasion, short disease-free time and poor overall survival. Tanaka, F.,et al., Cancer Res. 62 (2002) 7124-29 reviewed a total of 236 patientsof non-small cell lung cancer (NSCLC) with pathological stage-I to-IIIA, respectively. Using immunohistochemistry, they found that 16.9%of the NSCLC patients were ANG-2 positive. The microvessel density forANG-2 positive tumor is significantly higher than that of ANG-2negative. Such an angiogenic effect of ANG-2 was seen only when VEGFexpression was high. Moreover, positive expression of ANG-2 was asignificant factor to predict a poor postoperative survival. Tanaka, F.,et al., Cancer Res. 62 (2002) 7124-29. However, they found nosignificant correlation between Ang-1 expression and the microvesseldensity. Tanaka, F., et al., Cancer Res. 62 (2002) 7124-29. Theseresults suggest that ANG-2 is an indicator of poor prognosis patientswith several types of cancer.

Recently, using an ANG-2 knockout mouse model, Yancopoulos' groupreported that ANG-2 is required for postnatal angiogenesis. Gale, N. W.,et al., Dev. Cell 3 (2002) 411-23. They showed that the developmentallyprogrammed regression of the hyaloid vasculature in the eye does notoccur in the ANG-2 knockout mice and their retinal blood vessels fail tosprout out from the central retinal artery. Gale, N. W., et al., Dev.Cell 3 (2002) 411-23. They also found that deletion of ANG-2 results inprofound defects in the patterning and function of the lymphaticvasculature. Gale, N. W., et al., Dev. Cell 3 (2002) 411-23. Geneticrescue with Ang-1 corrects the lymphatic, but not the angiogenesisdefects. Gale, N. W., et al., Dev. Cell 3 (2002) 411-23.

Peters and his colleagues reported that soluble Tie2, when deliveredeither as recombinant protein or in a viral expression vector, inhibitedin vivo growth of murine mammary carcinoma and melanoma in mouse models.Lin, P., et al., Proc. Natl. Acad. Sci. USA 95 (1998) 8829-34; Lin, P.,et al., J. Clin. Invest. 100 (1997) 2072-78. Vascular densities in thetumor tissues so treated were greatly reduced. In addition, soluble Tie2blocked angiogenesis in the rat corneal stimulated by tumor cellconditioned media. Lin, P., et al., J. Clin. Invest. 100 (1997) 2072-78.Furthermore, Isner and his team demonstrated that addition of ANG-2 toVEGF promoted significantly longer and more circumferentialneovascularity than VEGF alone. Asahara, T., et al., Circ. Res., 83(1998) 233-40. Excess soluble Tie2 receptor precluded modulation ofVEGF-induced neovascularization by ANG-2. Asahara, T., et al., Circ.Res., 83, (1998) 233-40. Siemeister, G., et al., Cancer Res. 59 (1999)3185-91 showed with nude mouse xenografts that overexpression of theextracellular ligand-binding domains of either Flt-1 or Tie2 in thexenografts results in significant inhibition of pathway could not becompensated by the other one, suggesting that the VEGF receptor pathwayand the Tie2 pathway should be considered as two independent mediatorsessential for the process of in vivo angiogenesis. Siemeister, G., etal., Cancer Res. 59 (1999) 3185-91. This is proven by a more recentpublication by White, R. R., et al., Proc. Natl. Acad. Sci. USA 100(2003) 5028-33. In their study, it was demonstrated that anuclease-resistant RNA aptamer that specifically binds and inhibitsANG-2 significantly inhibited neovascularization induced by bFGF in therat corneal micropocket angiogenesis model.

SUMMARY OF THE INVENTION

The present invention relates to a method of treating a disease ordisorder in a patient comprising administering, to a patient in need ofsuch treatment, an antibody which binds specifically to humanangiopoietin-2 (ANG-2), wherein said antibody comprises:

-   -   (A) a heavy chain variable domain which comprises a CDR3 region        of SEQ ID NO: 1, a CDR2 region of SEQ ID NO: 2 and a CDR1 region        of SEQ ID NO: 3;    -   (B) a light chain variable domain which comprises a CDR3 region        of SEQ ID NO: 4, a CDR2 region of SEQ ID NO: 5 and a CDR1 region        of SEQ ID NO: 6.

The present invention also relates to a method for preventing metastasisin a patient suffering from primary cancer comprising administering, toa patient in need of such preventative treatment, an antibody whichbinds specifically to human angiopoietin-2 (ANG-2), wherein saidantibody comprises:

-   -   (A) a heavy chain variable domain which comprises a CDR3 region        of SEQ ID NO: 1, a CDR2 region of SEQ ID NO: 2 and a CDR1 region        of SEQ ID NO: 3;    -   (B) a light chain variable domain which comprises a CDR3 region        of SEQ ID NO: 4, a CDR2 region of SEQ ID NO: 5 and a CDR1 region        of SEQ ID NO: 6.

Description of the Amino Acid Sequences

-   SEQ ID NO: 1 heavy chain CDR3, <ANG-2>Ang2i_LC06-   SEQ ID NO: 2 heavy chain CDR2, <ANG-2>Ang2i_LC06-   SEQ ID NO: 3 heavy chain CDR1, <ANG-2>Ang2i_LC06-   SEQ ID NO: 4 light chain CDR3, <ANG-2>Ang2i_LC06-   SEQ ID NO: 5 light chain CDR2, <ANG-2>Ang2i_LC06-   SEQ ID NO: 6 light chain CDR1, <ANG-2>Ang2i_LC06-   SEQ ID NO: 7 heavy chain variable domain, <ANG-2>Ang2i_LC06-   SEQ ID NO: 8 light chain variable domain, <ANG-2>Ang2i_LC06-   SEQ ID NO: 9 heavy chain CDR3, <ANG-2>Ang2i_LC07-   SEQ ID NO: 10 heavy chain CDR2, <ANG-2>Ang2i_LC07-   SEQ ID NO: 11 heavy chain CDR1, <ANG-2>Ang2i_LC07-   SEQ ID NO: 12 light chain CDR3, <ANG-2>Ang2i_LC07-   SEQ ID NO: 13 light chain CDR2, <ANG-2>Ang2i_LC07-   SEQ ID NO: 14 light chain CDR1, <ANG-2>Ang2i_LC07-   SEQ ID NO: 15 heavy chain variable domain, <ANG-2>Ang2i_LC07-   SEQ ID NO: 16 light chain variable domain, <ANG-2>Ang2i_LC07-   SEQ ID NO: 17 heavy chain CDR3, <ANG-2>Ang2k_LC08-   SEQ ID NO: 18 heavy chain CDR2, <ANG-2>Ang2k_LC08-   SEQ ID NO: 19 heavy chain CDR1, <ANG-2>Ang2k_LC08-   SEQ ID NO: 20 light chain CDR3, <ANG-2>Ang2k_LC08-   SEQ ID NO: 21 light chain CDR2, <ANG-2>Ang2k_LC08-   SEQ ID NO: 22 light chain CDR1, <ANG-2>Ang2k_LC08-   SEQ ID NO: 23 heavy chain variable domain, <ANG-2>Ang2k_LC08-   SEQ ID NO: 24 light chain variable domain, <ANG-2>Ang2k_LC08-   SEQ ID NO: 25 heavy chain CDR3, <ANG-2>Ang2s_LC09-   SEQ ID NO: 26 heavy chain CDR2, <ANG-2>Ang2s_LC09-   SEQ ID NO: 27 heavy chain CDR1, <ANG-2>Ang2s_LC09-   SEQ ID NO: 28 light chain CDR3, <ANG-2>Ang2s_LC09-   SEQ ID NO: 29 light chain CDR2, <ANG-2>Ang2s_LC09-   SEQ ID NO: 30 light chain CDR1, <ANG-2>Ang2s_LC09-   SEQ ID NO: 31 heavy chain variable domain, <ANG-2>Ang2s_LC09-   SEQ ID NO: 32 light chain variable domain, <ANG-2>Ang2s_LC09-   SEQ ID NO: 33 heavy chain CDR3, <ANG-2>Ang2i_LC10-   SEQ ID NO: 34 heavy chain CDR2, <ANG-2>Ang2i_LC10-   SEQ ID NO: 35 heavy chain CDR1, <ANG-2>Ang2i_LC10-   SEQ ID NO: 36 light chain CDR3, <ANG-2>Ang2i_LC10-   SEQ ID NO: 37 light chain CDR2, <ANG-2>Ang2i_LC10-   SEQ ID NO: 38 light chain CDR1, <ANG-2>Ang2i_LC10-   SEQ ID NO: 39 heavy chain variable domain, <ANG-2>Ang2i_LC10-   SEQ ID NO: 40 light chain variable domain, <ANG-2>Ang2i_LC10-   SEQ ID NO: 41 heavy chain CDR3, <ANG-2>Ang2k_LC11-   SEQ ID NO: 42 heavy chain CDR2, <ANG-2>Ang2k_LC11-   SEQ ID NO: 43 heavy chain CDR1, <ANG-2>Ang2k_LC11-   SEQ ID NO: 44 light chain CDR3, <ANG-2>Ang2k_LC11-   SEQ ID NO: 45 light chain CDR2, <ANG-2>Ang2k_LC11-   SEQ ID NO: 46 light chain CDR1, <ANG-2>Ang2k_LC11-   SEQ ID NO: 47 heavy chain variable domain, <ANG-2>Ang2k_LC11-   SEQ ID NO: 48 light chain variable domain, <ANG-2>Ang2k_LC11-   SEQ ID NO: 49 heavy chain CDR3, <ANG-2>Ang2s_R3_LC03-   SEQ ID NO: 50 heavy chain CDR2, <ANG-2>Ang2s_R3_LC03-   SEQ ID NO: 51 heavy chain CDR1, <ANG-2>Ang2s_R3_LC03-   SEQ ID NO: 52 light chain CDR3, <ANG-2>Ang2s_R3_LC03-   SEQ ID NO: 53 light chain CDR2, <ANG-2>Ang2s_R3_LC03-   SEQ ID NO: 54 light chain CDR1, <ANG-2>Ang2s_R3_LC03-   SEQ ID NO: 55 heavy chain variable domain, <ANG-2>Ang2s_R3_LC03-   SEQ ID NO: 56 light chain variable domain, <ANG-2>Ang2s_R3_LC03-   SEQ ID NO: 57 human heavy chain constant region derived from IgG1-   SEQ ID NO: 58 human heavy chain constant region derived from IgG4-   SEQ ID NO: 59 kappa light chain constant region-   SEQ ID NO: 60 lambda light chain constant region-   SEQ ID NO: 61 Human Tie-2 receptor-   SEQ ID NO: 62 Human angiopoietin-2 (ANG-2) with leader and His-tag-   SEQ ID NO: 63 Human angiopoietin-1 (ANG-1) with leader and His-tag

DESCRIPTION OF THE FIGURES

FIG. 1 Cloning of IgGs for transient expressions into expression vectorstransient expressions A) Ang2i-LC06 (FIG. 1A) B.) Ang2i-LC06 (FIG. 1B)

FIG. 2 SDS-PAGE Gel of purified anti ANG-2 antibodies Ang2i-LC06,Ang2i-LC07 and Ang2k-LC08

FIG. 3 Angiopoietin-Tie2 interaction ELISA

FIG. 4 Inhibition of ANG-2 binding to Tie2 by Ang2i-LC06 and Ang2k-LC08

FIG. 5 Inhibition of ANG-1 binding to Tie2 by Ang2i-LC06 and Ang2k-LC08

FIG. 6 Colo205 xenograft model to test in vivo efficacy of anti ANG-2antibodies

FIG. 7 KPL-4 xenograft model to test in vivo efficacy of anti ANG-2antibodies.

FIG. 8 ANG-1 binding via Biacore sensogramm.

FIG. 9 Prevention of lung metastasis/secondary tumors by the antibodiesaccording to the invention in primary colon tumor xenograft (9A) andprimary breast xenograft (9B)

FIG. 10 Inhibition of tethinopaty by the antibodies according to theinvention.

DETAILED DESCRIPTION OF THE INVENTION

The invention comprises an antibody which binds specifically to humanangiopoietin-2 (ANG-2), wherein said antibody comprises, as a heavychain variable domain CDR3 region, a CDR3 region selected from the groupconsisting of: SEQ ID NO: 1, SEQ ID NO: 9, SEQ ID NO: 17, SEQ ID NO: 25,SEQ ID NO: 33, SEQ ID NO: 41, and SEQ ID NO: 49.

In one embodiment of the invention the antibody comprises:

-   a) a heavy chain variable domain which comprises:-   a CDR3 region selected from the group consisting of: SEQ ID NO: 1,    SEQ ID NO: 9, SEQ ID NO: 17, SEQ ID NO: 25, SEQ ID NO: 33, SEQ ID    NO: 41, and SEQ ID NO: 49;-   a CDR2 region selected from the group consisting of: SEQ ID NO: 2,    SEQ ID NO: 10, SEQ ID NO: 18, SEQ ID NO: 26, SEQ ID NO: 34, SEQ ID    NO: 42, and SEQ ID NO: 50; and-   a CDR1 region selected from the group consisting of: SEQ ID NO: 3,    SEQ ID NO: 11, SEQ ID NO: 19, SEQ ID NO: 27, SEQ ID NO: 35, SEQ ID    NO: 43, and SEQ ID NO: 51; and-   b) a light chain variable domain which comprises:-   a CDR3 region selected from the group consisting of: SEQ ID NO: 4,    SEQ ID NO: 12, SEQ ID NO: 20, SEQ ID NO: 28, SEQ ID NO: 36, SEQ ID    NO: 44, and SEQ ID NO: 52;-   a CDR2 region selected from the group consisting of: SEQ ID NO: 5,    SEQ ID NO: 13, SEQ ID NO: 21, SEQ ID NO: 29, SEQ ID NO: 37, SEQ ID    NO: 45, and SEQ ID NO: 53; and-   a CDR1 region selected from the group consisting of: SEQ ID NO: 6,    SEQ ID NO: 14, SEQ ID NO: 22, SEQ ID NO: 30, SEQ ID NO: 38, SEQ ID    NO: 46, and SEQ ID NO: 54.

Preferably the antibody comprises:

-   a) a heavy chain variable domain selected from the group consisting    of: SEQ ID NO: 7, SEQ ID NO: 15, SEQ ID NO: 23, SEQ ID NO: 31, SEQ    ID NO: 39, SEQ ID NO: 47, and SEQ ID NO: 55; and-   b) a light chain variable domain selected from the group consisting    of: SEQ ID NO: 8, SEQ ID NO: 16, SEQ ID NO: 24, SEQ ID NO: 32, SEQ    ID NO: 40, SEQ ID NO: 48, and SEQ ID NO: 56.

Another embodiment of the invention is an antibody which bindsspecifically to human ANG-2, which is characterized in that the antibodyis does not specifically bind to human Angiopoietin 1 (ANG-1). Typicalantibodies which specifically bind to human ANG-2, but not to humanANG-1 are e.g. Ang2s_R3_LC03, Ang2s_LC09, Ang2i_LC06, Ang2i_LC07, andantibodies binding to the same epitope as Ang2s_R3_LC03, Ang2s_LC09,Ang2i_LC06, Ang2i_LC07, and Ang2i_LC10. Preferred such antibodies arethose which bind to the same epitope as Ang2i_LC06. Therefore, in oneembodiment of the invention, the antibody binds specifically to humanangiopoietin-2 (ANG-2) but not to human ANG-1 binds to the same epitopeas Ang2s_R3_LC03, Ang2s_LC09, Ang2i_LC06, Ang2i_LC07, or Ang2i_LC10, andpreferably to the same epitope as Ang2i_LC06. Such antibodies bindspecifically to ANG-2, but not to ANG-1 can have improved propertiessuch as efficacy, less toxicity, pharmacokinetic properties compared toANG-2 and ANG-1 specific antibodies.

Therefore in one embodiment of the invention the antibody is one whichbinds specifically to human angiopoietin-2 (ANG-2) but not to humanANG-1 and comprises:

-   a) a heavy chain variable domain which comprises:-   a CDR3 region selected from the group consisting of: SEQ ID NO: 1,    SEQ ID NO: 9, SEQ ID NO: 25, SEQ ID NO: 33, and SEQ ID NO: 49;-   a CDR2 region selected from the group consisting of: SEQ ID NO: 2,    SEQ ID NO: 10, SEQ ID NO: 26, SEQ ID NO: 34, and SEQ ID NO: 50; and-   a CDR1 region selected from the group consisting of: SEQ ID NO: 3,    SEQ ID NO: 11, SEQ ID NO: 27, SEQ ID NO: 35, and SEQ ID NO: 51; and-   b) a light chain variable domain which comprises:-   a CDR3 region selected from the group consisting of: SEQ ID NO: 4,    SEQ ID NO: 12, SEQ ID NO: 28, SEQ ID NO: 36, and SEQ ID NO: 52;-   a CDR2 region selected from the group consisting of: SEQ ID NO: 5,    SEQ ID NO: 13, SEQ ID NO: 29, SEQ ID NO: 37, and SEQ ID NO: 53; and-   a CDR1 region selected from the group consisting of: SEQ ID NO: 6,    SEQ ID NO: 14, SEQ ID NO: 30, SEQ ID NO: 38, and SEQ ID NO: 54.

Preferably the antibody binds specifically to human angiopoietin-2(ANG-2) but not to human ANG-1 and comprises:

-   a) a heavy chain variable domain selected from the group consisting    of: SEQ ID NO: 7, SEQ ID NO: 15, SEQ ID NO: 31, SEQ ID NO: 39, and    SEQ ID NO: 55; and-   b) a light chain variable domain selected from the group consisting    of: SEQ ID NO: 8, SEQ ID NO: 16, SEQ ID NO: 32, SEQ ID NO: 40, and    SEQ ID NO: 56.

In one embodiment said antibody according to the invention comprises:

-   a) a heavy chain variable domain which comprises a CDR3 region of    SEQ ID NO: 1 or SEQ ID NO: 9, a CDR2 region of SEQ ID NO: 2 or SEQ    ID NO: 10, and a CDR1 region of SEQ ID NO: 3 or SEQ ID NO: 11, and-   b) a light chain variable domain which comprises a CDR3 region of    SEQ ID NO: 4 or SEQ ID NO: 12, a CDR2 region of SEQ ID NO: 5 or SEQ    ID NO: 13, and a CDR1 region of SEQ ID NO: 6 or SEQ ID NO: 14.

In one embodiment the antibody according to the invention comprises:

-   a) a heavy chain variable domain of SEQ ID NO: 7 or SEQ ID NO: 15;    and-   b) a light chain variable domain of SEQ ID NO: 8 or SEQ ID NO: 16.

In one embodiment the antibody according to the invention comprises:

-   a) a heavy chain variable domain which comprises a CDR3 region of    SEQ ID NO: 1, a CDR2 region of SEQ ID NO: 2, and a CDR1 region of    SEQ ID NO: 3, and-   b) a light chain variable domain which comprises a CDR3 region of    SEQ ID NO: 4, a CDR2 region of SEQ ID NO: 5, and a CDR1 region of    SEQ ID NO: 6.

In one embodiment the antibody according to the invention comprises:

-   a) a heavy chain variable domain of SEQ ID NO: 7; and-   b) a light chain variable domain of SEQ ID NO: 8.

In one embodiment the antibody according to the invention comprises:

-   a) a heavy chain variable domain which comprises a CDR3 region of    SEQ ID NO: 17, a CDR2 region of SEQ ID NO: 18, and a CDR1 region of    SEQ ID NO: 19, and-   b) a light chain variable domain which comprises a CDR3 region of    SEQ ID NO: 20, a CDR2 region of SEQ ID NO: 21, and a CDR1 region of    SEQ ID NO: 22.

In one embodiment the antibody according to the invention comprises:

-   a) a heavy chain variable domain of SEQ ID NO: 23; and-   b) a light chain variable domain of SEQ ID NO: 24.

Preferably the antibody according to the invention is of human IgG1subclass or is of human IgG4 subclass.

The term “antibody” encompasses the various forms of antibody structuresincluding but not being limited to whole antibodies and antibodyfragments, The antibody according to the invention is preferably ahumanized antibody, chimeric antibody, or further genetically engineeredantibody, as long as the characteristic properties according to theinvention are retained.

“Antibody fragments” comprise a portion of a full length antibody,preferably the variable domain thereof, or at least the antigen bindingsite thereof. Examples of antibody fragments include diabodies,single-chain antibody molecules (scFv or scFab), and multispecificantibodies (e.g. bispecific) formed from antibody fragments. scFvantibodies are, e.g. described in Houston, J. S., Methods in Enzymol.203 (1991) 46-88). In addition, antibody fragments comprise single chainpolypeptides having the characteristics of a V_(H) domain, namely beingable to assemble together with a V_(L) domain, or of a V_(L) domainbinding to ANG-2, namely being able to assemble together with a V_(H)domain to a functional antigen binding site and thereby providing theproperty. ScFvs can be stabilized using e.g. a) disulfide stabilization(see e.g. in WO 94/029350, Rajagopal, V., et al., Prot. Engin. (1997)1453-59; Kobayashi, H., et al., Nuclear Medicine & Biology, Vol. 25,(1998) 387-393; or Schmidt, M., et al., Oncogene (1999) 18 1711-1721.)or b) stabilized frameworks (e.g. by specific mutations of the see e.g.WO 2007/109254 specific stabilized frameworks see e.g. U.S. Pat. No.7,258,985, Furrer, F., et al., Invest. Ophthalmol. Vis. Sci. 50 (2009),pp. 771-778 or Ottiger, M., et al., Invest. Ophthalmol. Vis. Sci. 50(2009), pp. 779-786.

The terms “monoclonal antibody” or “monoclonal antibody composition” asused herein refer to a preparation of antibody molecules of a singleamino acid composition.

The term “chimeric antibody” refers to an antibody comprising a variableregion, i.e., binding region, from one source or species and at least aportion of a constant region derived from a different source or species,usually prepared by recombinant DNA techniques. Chimeric antibodiescomprising a murine variable region and a human constant region arepreferred. Other preferred forms of “chimeric antibodies” encompassed bythe present invention are those in which the constant region has beenmodified or changed from that of the original antibody to generate theproperties according to the invention, especially in regard to C1qbinding and/or Fc receptor (FcR) binding. Such chimeric antibodies arealso referred to as “class-switched antibodies.”. Chimeric antibodiesare the product of expressed immunoglobulin genes comprising DNAsegments encoding immunoglobulin variable regions and DNA segmentsencoding immunoglobulin constant regions. Methods for producing chimericantibodies involve conventional recombinant DNA and gene transfectiontechniques are well known in the art. See e.g. Morrison, S. L., et al.,Proc. Natl. Acad. Sci. USA 81 (1984) 6851-6855; U.S. Pat. Nos. 5,202,238and 5,204,244.

The term “humanized antibody” refers to antibodies in which theframework or “complementarity determining regions” (CDR) have beenmodified to comprise the CDR of an immunoglobulin of differentspecificity as compared to that of the parent immunoglobulin. In apreferred embodiment, a murine CDR is grafted into the framework regionof a human antibody to prepare the “humanized antibody.” See e.g.Riechmann, L., et al., Nature 332 (1988) 323-327; and Neuberger, M. S.,et al., Nature 314 (1985) 268-270. Particularly preferred CDRscorrespond to those representing sequences recognizing the antigensnoted above for chimeric antibodies. Other forms of “humanizedantibodies” encompassed by the present invention are those in which theconstant region has been additionally modified or changed from that ofthe original antibody to generate the properties according to theinvention, especially in regard to C1q binding and/or Fc receptor (FcR)binding.

The term “human antibody”, as used herein, is intended to includeantibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies are well-known in thestate of the art (van Dijk, M. A., and van de Winkel, J. G., Curr. Opin.Chem. Biol. 5 (2001) 368-374). Human antibodies can also be produced intransgenic animals (e.g., mice) that are capable, upon immunization, ofproducing a full repertoire or a selection of human antibodies in theabsence of endogenous immunoglobulin production. Transfer of the humangerm-line immunoglobulin gene array in such germ-line mutant mice willresult in the production of human antibodies upon antigen challenge(see, e.g., Jakobovits, A., et al., Proc. Natl. Acad. Sci. USA 90 (1993)2551-2555; Jakobovits, A., et al., Nature 362 (1993) 255-258;Brueggemann, M., et al., Year Immunol. 7 (1993) 33-40). Human antibodiescan also be produced in phage display libraries (Hoogenboom, H. R., andWinter, G., J. Mol. Biol. 227 (1992) 381-388; Marks, J. D., et al., J.Mol. Biol. 222 (1991) 581-597). The techniques of Cole, S. P. C., etal., and Boerner, et al. are also available for the preparation of humanmonoclonal antibodies (Cole, S. P. C., et al., Monoclonal Antibodies andCancer Therapy, Liss, A. R., (1985) 77-96; and Boerner, P., et al., J.Immunol. 147 (1991) 86-95). As already mentioned for chimeric andhumanized antibodies according to the invention the term “humanantibody” as used herein also comprises such antibodies which aremodified in the constant region to generate the properties according tothe invention, especially in regard to C1q binding and/or FcR binding,e.g. by “class switching” i.e. change or mutation of Fc parts (e.g. fromIgG1 to IgG4 and/or IgG1/IgG4 mutation.)

The term “recombinant human antibody”, as used herein, is intended toinclude all human antibodies that are prepared, expressed, created orisolated by recombinant means, such as antibodies isolated from a hostcell such as a NS0 or CHO cell or from an animal (e.g. a mouse) that istransgenic for human immunoglobulin genes or antibodies expressed usinga recombinant expression vector transfected into a host cell. Suchrecombinant human antibodies have variable and constant regions in arearranged form. The recombinant human antibodies according to theinvention have been subjected to in vivo somatic hypermutation. Thus,the amino acid sequences of the VH and VL regions of the recombinantantibodies are sequences that, while derived from and related to humangerm line VH and VL sequences, may not naturally exist within the humanantibody germ line repertoire in vivo.

The “variable domain” (variable domain of a light chain (V_(L)),variable domain of a heavy chain (V_(H))) as used herein denotes each ofthe pair of light and heavy chain domains which are involved directly inbinding the antibody to the antigen. The variable light and heavy chaindomains have the same general structure and each domain comprises fourframework (FR) regions whose sequences are widely conserved, connectedby three “hypervariable regions” (or complementary determining regions,CDRs). The framework regions adopt a β-sheet conformation and the CDRsmay form loops connecting the β-sheet structure. The CDRs in each chainare held in their three-dimensional structure by the framework regionsand form together with the CDRs from the other chain the antigen bindingsite. The antibody's heavy and light chain CDR3 regions play aparticularly important role in the binding specificity/affinity of theantibodies according to the invention and therefore provide a furtherobject of the invention.

The term “antigen-binding portion of an antibody” when used herein referto the amino acid residues of an antibody which are responsible forantigen-binding. The antigen-binding portion of an antibody comprisesamino acid residues from the “complementary determining regions” or“CDRs”. “Framework” or “FR” regions are those variable domain regionsother than the hypervariable region residues as herein defined.Therefore, the light and heavy chain variable domains of an antibodycomprise from N- to C-terminus the domains FR1, CDR1, FR2, CDR2, FR3,CDR3, and FR4. Especially, CDR3 of the heavy chain is the region whichcontributes most to antigen binding and defines the antibody'sproperties. CDR and FR regions are determined according to the standarddefinition of Kabat, E. A., et al., Sequences of Proteins ofImmunological Interest, 5th ed., Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991) and/or those residues from a“hypervariable loop.”

The terms “nucleic acid” or “nucleic acid molecule”, as used herein, areintended to include DNA molecules and RNA molecules. A nucleic acidmolecule may be single-stranded or double-stranded, but preferably isdouble-stranded DNA.

The term “amino acid” as used within this application denotes the groupof naturally occurring carboxy α-amino acids comprising alanine (threeletter code: ala, one letter code: A), arginine (arg, R), asparagine(asn, N), aspartic acid (asp, D), cysteine (cys, C), glutamine (gln, Q),glutamic acid (glu, E), glycine (gly, G), histidine (his, H), isoleucine(ile, I), leucine (leu, L), lysine (lys, K), methionine (met, M),phenylalanine (phe, F), proline (pro, P), serine (ser, S), threonine(thr, T), tryptophan (trp, W), tyrosine (tyr, Y), and valine (val, V).

A nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid. For example, DNA for apresequence or secretory leader is operable linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operable linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operable linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are colinear, and, in the caseof a secretory leader, contiguous and in reading frame. However,enhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice.

As used herein, the expressions “cell”, “cell line”, and “cell culture”are used interchangeably and all such designations include progeny.Thus, the words “transformants” and “transformed cells” include theprimary subject cell and cultures derived therefrom without regard forthe number of transfers. It is also understood that all progeny may notbe precisely identical in DNA content, due to deliberate or inadvertentmutations. Variant progeny that have the same function or biologicalactivity as screened for in the originally transformed cell areincluded.

As used herein, the terms “bind”, “binds”, and “binding” refer to thebinding of the antibody to an epitope of an antigen in an in vitroassay, preferably in an plasmon resonance assay (BIAcore, GE-HealthcareUppsala, Sweden) (Example 3) with purified wild-type ANG-2 antigen. Theaffinity of the binding is defined by the terms ka (rate constant forthe association of the antibody from the antibody/antigen complex),k_(D) (dissociation constant), and K_(D) (k_(D)/ka). Binding means abinding affinity (K_(D)) of 10⁻⁸ mol/l or less, preferably 10⁻⁹ M to10⁻¹³ mol/1.

Binding of the antibody to the FcγRIII can be investigated by a BIAcoreassay (GE-Healthcare Uppsala, Sweden). The affinity of the binding isdefined by the terms ka (rate constant for the association of theantibody from the antibody/antigen complex), k_(D) (dissociationconstant), and K_(D) (k_(D)/ka).

As used herein, the term “not binding to ANG-1” denotes that theantibody has an EC50-value above 8000 ng/ml in an in vitro ANG-1 bindingELISA assay (according to Example 2).

The term “epitope” includes any polypeptide determinant capable ofspecific binding to an antibody. In certain embodiments, epitopedeterminant include chemically active surface groupings of moleculessuch as amino acids, sugar side chains, phosphoryl, or sulfonyl, and, incertain embodiments, may have specific three dimensional structuralcharacteristics, and or specific charge characteristics. An epitope is aregion of an antigen that is bound by an antibody.

The “Fc part” of an antibody is not involved directly in binding of anantibody to an antigen, but exhibit various effector functions. A “Fcpart of an antibody” is a term well known to the skilled artisan anddefined on the basis of papain cleavage of antibodies. Depending on theamino acid sequence of the constant region of their heavy chains,antibodies or immunoglobulins are divided in the classes: IgA, IgD, IgE,IgG and IgM, and several of these may be further divided into subclasses(isotypes), e.g. IgG1, IgG2, IgG3, and IgG4, IgA1, and IgA2. Accordingto the heavy chain constant regions the different classes ofimmunoglobulins are called α, δ, ε, γ, and μ, respectively. The Fc partof an antibody is directly involved in ADCC (antibody-dependentcell-mediated cytotoxicity) and CDC (complement-dependent cytotoxicity)based on complement activation, C1q binding and Fc receptor binding.Complement activation (CDC) is initiated by binding of complement factorC1q to the Fc part of most IgG antibody subclasses. While the influenceof an antibody on the complement system is dependent on certainconditions, binding to C1q is caused by defined binding sites in the Fcpart. Such binding sites are known in the state of the art and describede.g. by Boakle, R. J., et al., Nature 282 (1975) 742-743, Lukas, T. J.,et al., J. Immunol. 127 (1981) 2555-2560, Brunhouse, R., and Cebra, J.J., Mol. Immunol. 16 (1979) 907-917, Burton, D. R., et al., Nature 288(1980) 338-344, Thommesen, J. E., et al., Mol. Immunol. 37 (2000)995-1004, Idusogie, E. E., et al., J. Immunol. 164 (2000) 4178-4184,Hezareh, M., et al., J. Virology 75 (2001) 12161-12168, Morgan, A., etal., Immunology 86 (1995) 319-324, EP 0307434. Such binding sites aree.g. L234, L235, D270, N297, E318, K320, K322, P331 and P329 (numberingaccording to EU index of Kabat, see below). Antibodies of subclass IgG1,IgG2 and IgG3 usually show complement activation and C1q and C3 binding,whereas IgG4 do not activate the complement system and do not bind C1qand C3.

The antibody according to the invention preferably comprises a Fc partfrom human origin which is Fc part of a human antibody of the subclassIgG1.

The antibody according to the invention is characterized in that theconstant chains are of human origin. Such constant chains are well knownin the state of the art and e.g. described by Kabat, E. A. (see e.g.Johnson, G. and Wu, T. T., Nucleic Acids Res. 28 (2000) 214-218). Forexample, a useful human heavy chain constant region comprises an aminoacid sequence of SEQ ID NO: 57 or of SEQ ID NO: 58. For example, auseful human light chain constant region comprises an amino acidsequence of a kappa-light chain constant region of SEQ ID NO: 59, or ofa lambda-light chain constant region of SEQ ID NO: 60.

The term “constant region” as used within the current applicationsdenotes the sum of the domains of an antibody other than the variableregion. The constant region is not involved directly in binding of anantigen, but exhibit various effector functions. Depending on the aminoacid sequence of the constant region of their heavy chains, antibodiesare divided in the classes: IgA, IgD, IgE, IgG and IgM, and several ofthese may be further divided into subclasses, such as IgG1, IgG2, IgG3,and IgG4, IgA1 and IgA2. The heavy chain constant regions thatcorrespond to the different classes of antibodies are called α, δ, ε, γ,and μ, respectively. The light chain constant regions which can be foundin all five antibody classes are called κ (kappa) and λ, (lambda).

The term “constant region derived from human origin” as used in thecurrent application denotes a constant heavy chain region of a humanantibody of the subclass IgG1, IgG2, IgG3, or IgG4 and/or a constantlight chain κ region. Such constant regions are well known in the stateof the art and e.g. described by Kabat, E. A., (see e.g. Johnson, G. andWu, T. T., Nucleic Acids Res. 28 (2000) 214-218; Kabat, E. A., et al.,Proc. Natl. Acad. Sci. USA 72 (1975) 2785-2788).

While antibodies of the IgG4 subclass show reduced Fc receptor(FcγRIIIa) binding, antibodies of other IgG subclasses show strongbinding. However Pro238, Asp265, Asp270, Asn297 (loss of Fccarbohydrate), Pro329, Leu234, Leu235, Gly236, Gly237, Ile253, Ser254,Lys288, Thr307, Gln311, Asn434, and His435 are residues which, ifaltered, provide also reduced Fc receptor binding (Shields, R. L., etal., J. Biol. Chem. 276 (2001) 6591-6604; Lund, J., et al., FASEB J. 9(1995) 115-119; Morgan, A., et al., Immunology 86 (1995) 319-324; EP 0307 434).

In one embodiment an antibody according to the invention has a reducedFcR binding compared to an IgG1 antibody and the monospecific bivalentparent antibody is in regard to FcR binding of IgG4 subclass or of IgG1or IgG2 subclass with a mutation in 5228, L234, L235 and/or D265, and/orcontains the PVA236 mutation. In one embodiment the mutations in themonospecific bivalent parent antibody are S228P, L234A, L235A, L235Eand/or PVA236. In another embodiment the mutations in the monospecificbivalent parent antibody are in IgG4 S228P and in IgG1 L234A and L235A.Constant heavy chain regions shown in SEQ ID NO: 57 and 58. In oneembodiment the constant heavy chain region of the monospecific bivalentparent antibody is of SEQ ID NO: 57 with mutations L234A and L235A. Inanother embodiment the constant heavy chain region of the monospecificbivalent parent antibody is of SEQ ID NO: 58 with mutation S228P. Inanother embodiment the constant light chain region of the monospecificbivalent parent antibody is a kappa light chain region of SEQ ID NO: 59,or a lambda light chain constant region of SEQ ID NO: 60. In oneembodiment of the invention the constant heavy chain region of themonospecific bivalent parent antibody is of SEQ ID NO: 57 or of SEQ IDNO: 58 with mutation S228P.

The constant region of an antibody is directly involved in ADCC(antibody-dependent cell-mediated cytotoxicity) and CDC(complement-dependent cytotoxicity). Complement activation (CDC) isinitiated by binding of complement factor C1q to the constant region ofmost IgG antibody subclasses. Binding of C1q to an antibody is caused bydefined protein-protein interactions at the so called binding site. Suchconstant region binding sites are known in the state of the art anddescribed e.g. by Lukas, T. J., et al., J. Immunol. 127 (1981)2555-2560; Brunhouse, R. and Cebra, J. J., Mol. Immunol. 16 (1979)907-917; Burton, D. R., et al., Nature 288 (1980) 338-344; Thommesen, J.E., et al., Mol. Immunol. 37 (2000) 995-1004; Idusogie, E. E., et al.,J. Immunol. 164 (2000) 4178-4184; Hezareh, M., et al., J. Virol. 75(2001) 12161-12168; Morgan, A., et al., Immunology 86 (1995) 319-324;and EP 0 307 434. Such constant region binding sites are, e.g.,characterized by the amino acids L234, L235, D270, N297, E318, K320,K322, P331, and P329 (numbering according to EU index of Kabat).

The term “antibody-dependent cellular cytotoxicity (ADCC)” refers tolysis of human target cells by an antibody according to the invention inthe presence of effector cells. ADCC is measured preferably by thetreatment of a preparation of CCR5 expressing cells with an antibodyaccording to the invention in the presence of effector cells such asfreshly isolated PBMC or purified effector cells from buffy coats, likemonocytes or natural killer (NK) cells or a permanently growing NK cellline.

The term “complement-dependent cytotoxicity (CDC)” denotes a processinitiated by binding of complement factor C1q to the Fc part of most IgGantibody subclasses. Binding of C1q to an antibody is caused by definedprotein-protein interactions at the so called binding site. Such Fc partbinding sites are known in the state of the art (see above). Such Fcpart binding sites are, e.g., characterized by the amino acids L234,L235, D270, N297, E318, K320, K322, P331, and P329 (numbering accordingto EU index of Kabat). Antibodies of subclass IgG1, IgG2, and IgG3usually show complement activation including C1q and C3 binding, whereasIgG4 does not activate the complement system and does not bind C1qand/or C3.

The antibody according to the invention is produced by recombinantmeans. Thus, one aspect of the current invention is a nucleic acidencoding the antibody according to the invention and a further aspect isa cell comprising said nucleic acid encoding an antibody according tothe invention. Methods for recombinant production are widely known inthe state of the art and comprise protein expression in prokaryotic andeukaryotic cells with subsequent isolation of the antibody and usuallypurification to a pharmaceutically acceptable purity. For the expressionof the antibodies as aforementioned in a host cell, nucleic acidsencoding the respective modified light and heavy chains are insertedinto expression vectors by standard methods. Expression is performed inappropriate prokaryotic or eukaryotic host cells like CHO cells, NS0cells, SP2/0 cells, HEK293 cells, COS cells, PER.C6 cells, yeast, or E.coli cells, and the antibody is recovered from the cells (supernatant orcells after lysis). General methods for recombinant production ofantibodies are well-known in the state of the art and described, forexample, in the review articles of Makrides, S. C., Protein Expr. Purif.17 (1999) 183-202; Geisse, S., et al., Protein Expr. Purif. 8 (1996)271-282; Kaufman, R. J., Mol. Biotechnol. 16 (2000) 151-161; Werner, R.G., J. Drug Res. 48 (1998) 870-880.

The antibodies according to the invention are suitably separated fromthe culture medium by conventional immunoglobulin purificationprocedures such as, for example, protein A-Sepharose, hydroxylapatitechromatography, gel electrophoresis, dialysis, or affinitychromatography. DNA and RNA encoding the monoclonal antibodies isreadily isolated and sequenced using conventional procedures. Thehybridoma cells can serve as a source of such DNA and RNA. Onceisolated, the DNA may be inserted into expression vectors, which arethen transfected into host cells such as HEK 293 cells, CHO cells, ormyeloma cells that do not otherwise produce immunoglobulin protein, toobtain the synthesis of recombinant monoclonal antibodies in the hostcells.

Amino acid sequence variants (or mutants) of the antibody according tothe invention are prepared by introducing appropriate nucleotide changesinto the antibody DNA, or by nucleotide synthesis. Such modificationscan be performed, however, only in a very limited range, e.g. asdescribed above. For example, the modifications do not alter the abovementioned antibody characteristics such as the IgG isotype and antigenbinding, but may improve the yield of the recombinant production,protein stability or facilitate the purification.

The term “host cell” as used in the current application denotes any kindof cellular system which can be engineered to generate the antibodiesaccording to the current invention. In one embodiment HEK293 cells andCHO cells are used as host cells. As used herein, the expressions“cell,” “cell line,” and “cell culture” are used interchangeably and allsuch designations include progeny. Thus, the words “transformants” and“transformed cells” include the primary subject cell and culturesderived therefrom without regard for the number of transfers. It is alsounderstood that all progeny may not be precisely identical in DNAcontent, due to deliberate or inadvertent mutations. Variant progenythat have the same function or biological activity as screened for inthe originally transformed cell are included.

Expression in NS0 cells is described by, e.g., Barnes, L. M., et al.,Cytotechnology 32 (2000) 109-123; Barnes, L. M., et al., Biotech.Bioeng. 73 (2001) 261-270. Transient expression is described by, e.g.,Durocher, Y., et al., Nucl. Acids. Res. 30 (2002) E9. Cloning ofvariable domains is described by Orlandi, R., et al., Proc. Natl. Acad.Sci. USA 86 (1989) 3833-3837; Carter, P., et al., Proc. Natl. Acad. Sci.USA 89 (1992) 4285-4289; and Norderhaug, L., et al., J. Immunol. Methods204 (1997) 77-87. A preferred transient expression system (HEK 293) isdescribed by Schlaeger, E.-J., and Christensen, K., in Cytotechnology 30(1999) 71-83 and by Schlaeger, E.-J., in J. Immunol. Methods 194 (1996)191-199.

The control sequences that are suitable for prokaryotes, for example,include a promoter, optionally an operator sequence, and a ribosomebinding site. Eukaryotic cells are known to utilize promoters, enhancersand polyadenylation signals.

A nucleic acid is “operably linked” when it is placed in a functionalrelationship with another nucleic acid sequence. For example, DNA for apre-sequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a pre-protein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are contiguous, and, in thecase of a secretory leader, contiguous and in reading frame. However,enhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice.

Purification of antibodies is performed in order to eliminate cellularcomponents or other contaminants, e.g. other cellular nucleic acids orproteins, by standard techniques, including alkaline/SDS treatment, CsClbanding, column chromatography, agarose gel electrophoresis, and otherswell known in the art. See Ausubel, F., et al., ed. Current Protocols inMolecular Biology, Greene Publishing and Wiley Interscience, New York(1987). Different methods are well established and widespread used forprotein purification, such as affinity chromatography with microbialproteins (e.g. protein A or protein G affinity chromatography), ionexchange chromatography (e.g. cation exchange (carboxymethyl resins),anion exchange (amino ethyl resins) and mixed-mode exchange), thiophilicadsorption (e.g. with beta-mercaptoethanol and other SH ligands),hydrophobic interaction or aromatic adsorption chromatography (e.g. withphenyl-sepharose, aza-arenophilic resins, or m-aminophenylboronic acid),metal chelate affinity chromatography (e.g. with Ni(II)- andCu(II)-affinity material), size exclusion chromatography, andelectrophoretical methods (such as gel electrophoresis, capillaryelectrophoresis) (Vijayalakshmi, M. A. Appl. Biochem. Biotech. 75 (1998)93-102).

The invention comprises a method for the treatment of a patient in needof therapy, characterized by administering to the patient atherapeutically effective amount of an antibody according to theinvention.

The invention comprises the use of an antibody according to theinvention for therapy.

The invention comprises the use of an antibody according to theinvention for the preparation of a medicament for the prevention ofmetastasis.

The invention comprises the use of an antibody according to theinvention for the preparation of a medicament for the treatment ofcancer.

One aspect of the invention is a pharmaceutical composition comprisingan antibody according to the invention. Another aspect of the inventionis the use of an antibody according to the invention for the manufactureof a pharmaceutical composition. A further aspect of the invention is amethod for the manufacture of a pharmaceutical composition comprising anantibody according to the invention. In another aspect, the presentinvention provides a composition, e.g. a pharmaceutical composition,containing an antibody according to the present invention, formulatedtogether with a pharmaceutical carrier.

Another aspect of the invention is said pharmaceutical composition forthe prevention of metastasis.

Another aspect of the invention is an antibody according to theinvention for the prevention of metastasis.

Another aspect of the invention is the use of an antibody according tothe invention for the manufacture of a medicament for the prevention ofmetastasis.

Another aspect of the invention is a method of prevention metastasis inpatient suffering from primary cancer by administering an antibodyaccording to the invention to a patient in the need of such preventativetreatment.

We could show highly efficient prevention of spotanouenesmetastasis/secondary tumors in vivo in a orthotopic and a subcutanouescancer model (see Example 9) (in contrast to experimental model wherethe tumor cells are injected i.v. This is similar to the clinicalsituation wherein cells disseminate from a primary tumor and metastaseto secondary organ like lung or liver (where secondary tumors).

The term “metastasis” according to the invention refers to thetransmission of cancerous cells from the primary tumor to one or moresites elsewhere in a patient where then secondary tumors develop.MetastasMeans to determine if a cancer has metastasized are known in theart and include bone scan, chest X-ray, CAT scan, MRI scan, and tumormarker tests.

The term “prevention of metastasis” or “prevention of secondary tumors”as used herein have the same meaning and refers a prophylactic agentagainst metastasis in patient suffering from relapsed HER2 positivecancer in this way inhibiting or reducing a further transmission ofcancerous cells from the primary tumor to one or more sites elsewhere ina patient. This means that the metastasis of the primary, tumor orcancer is prevented, delayed, or reduced and thus the development ofsecondary tumors is prevented, delayed, or reduced. Preferably themetastasis i.e secondary tumors of the lung are prevented or reduced,which means that metastatic transmission of cancerous cells from theprimary tumor to the lung is prevented or reduced.

Another aspect of the invention is said pharmaceutical composition forthe treatment of cancer.

Another aspect of the invention is an antibody according to theinvention for the treatment of cancer.

Another aspect of the invention is the use of an antibody according tothe invention for the manufacture of a medicament for the treatment ofcancer.

Another aspect of the invention is method of treatment of patientsuffering from cancer by administering an antibody according to theinvention to a patient in the need of such treatment.

As used herein, “pharmaceutical carrier” includes any and all solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents, and the like that arephysiologically compatible. Preferably, the carrier is suitable forintravenous, intramuscular, subcutaneous, parenteral, spinal orepidermal administration (e.g. by injection or infusion).

A composition of the present invention can be administered by a varietyof methods known in the art. As will be appreciated by the skilledartisan, the route and/or mode of administration will vary dependingupon the desired results. To administer a compound of the invention bycertain routes of administration, it may be necessary to coat thecompound with, or co-administer the compound with, a material to preventits inactivation. For example, the compound may be administered to asubject in an appropriate carrier, for example, liposomes, or a diluent.Pharmaceutically acceptable diluents include saline and aqueous buffersolutions. Pharmaceutical carriers include sterile aqueous solutions ordispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersion. The use of such media andagents for pharmaceutically active substances is known in the art.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intra-arterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal, epidural and intrasternal injection andinfusion.

The term cancer as used herein refers to proliferative diseases, such aslymphomas, lymphocytic leukemias, lung cancer, non small cell lung(NSCL) cancer, bronchioloalviolar cell lung cancer, bone cancer,pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous orintraocular melanoma, uterine cancer, ovarian cancer, rectal cancer,cancer of the anal region, stomach cancer, gastric cancer, colon cancer,breast cancer, uterine cancer, carcinoma of the fallopian tubes,carcinoma of the endometrium, carcinoma of the cervix, carcinoma of thevagina, carcinoma of the vulva, Hodgkin's Disease, cancer of theesophagus, cancer of the small intestine, cancer of the endocrinesystem, cancer of the thyroid gland, cancer of the parathyroid gland,cancer of the adrenal gland, sarcoma of soft tissue, cancer of theurethra, cancer of the penis, prostate cancer, cancer of the bladder,cancer of the kidney or ureter, renal cell carcinoma, carcinoma of therenal pelvis, mesothelioma, hepatocellular cancer, biliary cancer,neoplasms of the central nervous system (CNS), spinal axis tumors, brainstem glioma, glioblastoma multiforme, astrocytomas, schwanomas,ependymonas, medulloblastomas, meningiomas, squamous cell carcinomas,pituitary adenoma and Ewings sarcoma, including refractory versions ofany of the above cancers, or a combination of one or more of the abovecancers.

Another aspect of the invention is said pharmaceutical composition asanti-angiogenic agent. Such anti-angiogenic agent can be used for thetreatment of cancer, especially solid tumors, and other vasculardiseases.

Another aspect of the invention is the use of an antibody according tothe invention for the manufacture of a medicament for the treatment ofvascular diseases.

Another aspect of the invention is an antibody according to theinvention for the treatment of vascular diseases.

A preferred embodiment is an antibody according to the invention for thetreatment of retinopathy.

A preferred embodiment is the use of an antibody according to theinvention for the manufacture of a medicament for the treatment ofretinopathy

Another aspect of the invention is method of treatment of patientsuffering from vascular diseases by administering an antibody accordingto the invention to a patient in the need of such treatment.

The term “vascular diseases” includes Cancer, Inflammatory diseases,Atherosclerosis, Ischemia, Trauma, Sepsis, COPD, Asthma, Diabetes, AMD,Retinopathy, Stroke, Adipositas, Acute lung injury, Hemorrhage, Vascularleak e.g. Cytokine induced, Allergy, Graves' Disease, Hashimoto'sAutoimmune Thyroiditis, Idiopathic Thrombocytopenic Purpura, Giant CellArteritis, Rheumatoid Arthritis, Systemic Lupus Erythematosus (SLE),Lupus Nephritis, Crohn's Disease, Multiple Sclerosis, UlcerativeColitis, especially to solid tumors, intraocular neovascular syndromes(such as proliferative retinopathies or age-related macular degeneration(AMD)), rheumatoid arthritis, and psoriasis (Folkman, J., et al., J.Biol. Chem. 267 (1992) 10931-10934; Klagsbrun, M., et al., Annu Rev.Physiol. 53 (1991) 217-239; and Garner, A., Vascular diseases, In:Pathobiology of ocular disease, A dynamic approach, Garner, A., andKlintworth, G. K. (eds.), 2nd edition, Marcel Dekker, New York (1994),pp 1625-1710).

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofpresence of microorganisms may be ensured both by sterilizationprocedures, supra, and by the inclusion of various antibacterial andantifungal agents, for example, paraben, chlorobutanol, phenol, sorbicacid, and the like. It may also be desirable to include isotonic agents,such as sugars, sodium chloride, and the like into the compositions. Inaddition, prolonged absorption of the injectable pharmaceutical form maybe brought about by the inclusion of agents which delay absorption suchas aluminum monostearate and gelatin.

Regardless of the route of administration selected, the compounds of thepresent invention, which may be used in a suitable hydrated form, and/orthe pharmaceutical compositions of the present invention, are formulatedinto pharmaceutically acceptable dosage forms by conventional methodsknown to those of skill in the art.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of the present invention may be varied so as to obtain anamount of the active ingredient which is effective to achieve thedesired therapeutic response for a particular patient, composition, andmode of administration, without being toxic to the patient. The selecteddosage level will depend upon a variety of pharmacokinetic factorsincluding the activity of the particular compositions of the presentinvention employed, the route of administration, the time ofadministration, the rate of excretion of the particular compound beingemployed, the duration of the treatment, other drugs, compounds and/ormaterials used in combination with the particular compositions employed,the age, sex, weight, condition, general health and prior medicalhistory of the patient being treated, and like factors well known in themedical arts.

The composition must be sterile and fluid to the extent that thecomposition is deliverable by syringe. In addition to water, the carrierpreferably is an isotonic buffered saline solution.

Proper fluidity can be maintained, for example, by use of coating suchas lecithin, by maintenance of required particle size in the case ofdispersion and by use of surfactants. In many cases, it is preferable toinclude isotonic agents, for example, sugars, polyalcohols such asmannitol or sorbitol, and sodium chloride in the composition.

As used herein, the expressions “cell,” “cell line,” and “cell culture”are used interchangeably and all such designations include progeny.Thus, the words “transformants” and “transformed cells” include theprimary subject cell and cultures derived therefrom without regard forthe number of transfers. It is also understood that all progeny may notbe precisely identical in DNA content, due to deliberate or inadvertentmutations. Variant progeny that have the same function or biologicalactivity as screened for in the originally transformed cell areincluded. Where distinct designations are intended, it will be clearfrom the context.

The term “transformation” as used herein refers to process of transferof a vectors/nucleic acid into a host cell. If cells without formidablecell wall barriers are used as host cells, transfection is carried oute.g. by the calcium phosphate precipitation method as described byGraham, F. L., and van der Eb, Virology 52 (1973) 456-467. However,other methods for introducing DNA into cells such as by nuclearinjection or by protoplast fusion may also be used. If prokaryotic cellsor cells which contain substantial cell wall constructions are used,e.g. one method of transfection is calcium treatment using calciumchloride as described by Cohen, F. N, et al, PNAS. 69 (1972) 7110ff.

As used herein, “expression” refers to the process by which a nucleicacid is transcribed into mRNA and/or to the process by which thetranscribed mRNA (also referred to as transcript) is subsequently beingtranslated into peptides, polypeptides, or proteins. The transcripts andthe encoded polypeptides are collectively referred to as gene product.If the polynucleotide is derived from genomic DNA, expression in aeukaryotic cell may include splicing of the mRNA.

A “vector” is a nucleic acid molecule, in particular self-replicating,which transfers an inserted nucleic acid molecule into and/or betweenhost cells. The term includes vectors that function primarily forinsertion of DNA or RNA into a cell (e.g., chromosomal integration),replication of vectors that function primarily for the replication ofDNA or RNA, and expression vectors that function for transcriptionand/or translation of the DNA or RNA. Also included are vectors thatprovide more than one of the functions as described.

An “expression vector” is a polynucleotide which, when introduced intoan appropriate host cell, can be transcribed and translated into apolypeptide. An “expression system” usually refers to a suitable hostcell comprised of an expression vector that can function to yield adesired expression product.

The following examples, sequence listing and figures are provided to aidthe understanding of the present invention, the true scope of which isset forth in the appended claims. It is understood that modificationscan be made in the procedures set forth without departing from thespirit of the invention.

EXPERIMENTAL PROCEDURE 1

Materials & General Methods

General information regarding the nucleotide sequences of humanimmunoglobulins light and heavy chains is given in: Kabat, E. A., etal., Sequences of Proteins of Immunological Interest, 5th ed., PublicHealth Service, National Institutes of Health, Bethesda, Md. (1991).Amino acids of antibody chains are numbered and referred to according toEU numbering (Edelman, G. M., et al., Proc. Natl. Acad. Sci. USA 63(1969) 78-85; Kabat, E. A., et al., Sequences of Proteins ofImmunological Interest, 5th ed., Public Health Service, NationalInstitutes of Health, Bethesda, Md., (1991)).

Recombinant DNA Techniques

Standard methods were used to manipulate DNA as described in Sambrook,J. et al., Molecular cloning: A laboratory manual; Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989. The molecularbiological reagents were used according to the manufacturer'sinstructions.

Gene Synthesis

Desired gene segments were prepared from oligonucleotides made bychemical synthesis. The gene segments, which are flanked by singularrestriction endonuclease cleavage sites, were assembled by annealing andligation of oligonucleotides including PCR amplification andsubsequently cloned via the indicated restriction sites e.g. KpnI/Sad orAscI/PacI into a pPCRScript (Stratagene) based pGA4 cloning vector. TheDNA sequences of the subcloned gene fragments were confirmed by DNAsequencing. Gene synthesis fragments were ordered according to givenspecifications at Geneart (Regensburg, Germany).

DNA Sequence Determination

DNA sequences were determined by double strand sequencing performed atMediGenomix GmbH (Martinsried, Germany) or Sequiserve GmbH(Vaterstetten, Germany).

DNA and Protein Sequence Analysis and Sequence Data Management

The GCG's (Genetics Computer Group, Madison, Wis.) software packageversion 10.2 and Infomax's Vector NT1 Advance suite version 8.0 was usedfor sequence creation, mapping, analysis, annotation and illustration.

Expression Vectors

For the expression of the described antibodies variants of expressionplasmids for transient expression (e.g. in HEK293 EBNA or HEK293-Fcells) or for stable expression (e.g. in CHO cells) based either on acDNA organization with a CMV-Intron A promoter or on a genomicorganization with a CMV promoter (e.g. FIG. 1) were applied.

Beside the antibody expression cassette the vectors contained:

-   -   an origin of replication which allows replication of this        plasmid in E. coli, and    -   a β-lactamase gene which confers ampicillin resistance in E.        coli.

The transcription unit of the antibody gene is composed of the followingelements:

-   -   unique restriction site(s) at the 5′ end    -   the immediate early enhancer and promoter from the human        cytomegalovirus,    -   followed by the Intron A sequence in the case of the cDNA        organization,    -   a 5′-untranslated region of a human antibody gene,    -   a immunoglobulin heavy chain signal sequence,    -   the human antibody chain (heavy chain, modified heavy chain or        light chain) either as cDNA or as genomic organization with an        the immunoglobulin exon-intron organization    -   a 3′ untranslated region with a polyadenylation signal sequence,        and    -   unique restriction site(s) at the 3′ end.

The fusion genes comprising the heavy chain sequences of the selectedantibody as described below were generated by PCR and/or gene synthesisand assembled with known recombinant methods and techniques byconnection of the according nucleic acid segments e.g. using unique NsiIand EcoRI sites in the genomic heavy chain vectors. The subclonednucleic acid sequences were verified by DNA sequencing. For transientand stable transfections larger quantities of the plasmids were preparedby plasmid preparation from transformed E. coli cultures (Nucleobond AX,Macherey-Nagel).

Cell Culture Techniques

Standard cell culture techniques were used as described in CurrentProtocols in Cell Biology (2000), Bonifacino, J. S., Dasso, M., Harford,J. B., Lippincott-Schwartz, J. and Yamada, K. M. (eds.), John Wiley &Sons, Inc.

Transient Transfections in HEK293-F System

Antibodies were generated by transient transfection of the two plasmidsencoding the heavy or modified heavy chain, respectively and thecorresponding light chain using the HEK293-F system (Invitrogen)according to the manufacturer's instruction. Briefly, HEK293-F cells(Invitrogen) growing in suspension either in a shake flask or in astirred fermenter in serumfree FreeStyle 293 expression medium(Invitrogen) were transfected with a mix of the two respectiveexpression plasmids and 293fectin or fectin (Invitrogen). For e.g. 2 Lshake flask (Corning) HEK293-F cells were seeded at a density of 1.0E*6cells/mL in 600 mL and incubated at 120 rpm, 8% CO2. The day after thecells were transfected at a cell density of ca. 1.5E*6 cells/mL with ca.42 mL mix of A) 20 mL Opti-MEM (Invitrogen) with 600 μg total plasmidDNA (1 μg/mL) encoding the heavy or modified heavy chain, respectivelyand the corresponding light chain in an equimolar ratio and B) 20 mlOpti-MEM+1.2 mL 293 fectin or fectin (2 μl/mL). According to the glucoseconsumption glucose solution was added during the course of thefermentation. The supernatant containing the secreted antibody washarvested after 5-10 days and antibodies were either directly purifiedfrom the supernatant or the supernatant was frozen and stored.

Protein Determination

The protein concentration of purified antibodies and derivatives wasdetermined by determining the optical density (OD) at 280 nm, using themolar extinction coefficient calculated on the basis of the amino acidsequence according to Pace, C. N., et. al., Protein Science, 4 (1995),2411-1423.

Antibody Concentration Determination in Supernatants

The concentration of antibodies and derivatives in cell culturesupernatants was estimated by immunoprecipitation with Protein AAgarose-beads (Roche). 60 μL Protein A Agarose beads are washed threetimes in TBS-NP40 (50 mM Tris, pH 7.5, 150 mM NaCl, 1% Nonidet-P40).Subsequently, 1-15 mL cell culture supernatant are applied to theProtein A Agarose beads pre-equilibrated in TBS-NP40. After incubationfor at 1 h at room temperature the beads are washed on anUltrafree-MC-filter column (Amicon) once with 0.5 mL TBS-NP40, twicewith 0.5 mL 2× phosphate buffered saline (2×PBS, Roche) and briefly fourtimes with 0.5 mL 100 mM Na-citrate pH 5.0. Bound antibody is eluted byaddition of 35 μl NuPAGE® LDS Sample Buffer (Invitrogen). Half of thesample is combined with NuPAGE® Sample Reducing Agent or left unreduced,respectively, and heated for 10 min at 70° C. Consequently, 20 μl areapplied to an 4-12% NuPAGE® Bis-Tris SDS-PAGE (Invitrogen) (with MOPSbuffer for non-reduced SDS-PAGE and MES buffer with NuPAGE® Antioxidantrunning buffer additive (Invitrogen) for reduced SDS-PAGE) and stainedwith Coomassie Blue.

The concentration of antibodies and derivatives in cell culturesupernatants was measured by Protein A-HPLC chromatography. Briefly,cell culture supernatants containing antibodies and derivatives thatbind to Protein A were applied to a HiTrap Protein A column (GEHealthcare) in 50 mM K2HPO4, 300 mM NaCl, pH 7.3 and eluted from thematrix with 50 mM acetic acid, pH 2.5 on a Dionex HPLC-System. Theeluted protein was quantified by UV absorbance and integration of peakareas. A purified standard IgG1 antibody served as a standard.

Alternatively, the concentration of antibodies and derivatives in cellculture supernatants was measured by Sandwich-IgG-ELISA. Briefly,StreptaWell High Bind Strepatavidin A-96 well microtiter plates (Roche)were coated with 100 μL/well biotinylated anti-human IgG capturemolecule F(ab′)2<h-Fcγ>BI (Dianova) at 0.1 μg/mL for 1 h at roomtemperature or alternatively over night at 4° C. and subsequently washedthree times with 200 μL/well PBS, 0.05% Tween (PBST, Sigma). 100 μL/wellof a dilution series in PBS (Sigma) of the respective antibodycontaining cell culture supernatants was added to the wells andincubated for 1-2 h on a microtiterplate shaker at room temperature. Thewells were washed three times with 200 μL/well PBST and bound antibodywas detected with 100 μl F(ab′)2<hFcgamma>POD (Dianova) at 0.1 μg/mL asdetection antibody for 1-2 h on a microtiterplate shaker at roomtemperature. Unbound detection antibody was washed away three times with200 μL/well PBST and the bound detection antibody was detected byaddition of 100 μL ABTS/well. Determination of absorbance was performedon a Tecan Fluor Spectrometer at a measurement wavelength of 405 nm(reference wavelength 492 nm).

Protein Purification

Proteins were purified from filtered cell culture supernatants referringto standard protocols. In brief, antibodies were applied to a Protein ASepharose column (GE Healthcare) and washed with PBS. Elution ofantibodies was achieved at acidic pH followed by immediateneutralization of the sample. Aggregated protein was separated frommonomeric antibodies by size exclusion chromatography (Superdex 200, GEHealthcare) in 20 mM Histidine, 140 mM NaCl pH 6.0. Monomeric antibodyfractions were pooled, concentrated if required using e.g. a MILLIPOREAmicon Ultra (30 MWCO) centrifugal concentrator and stored at −80° C.Part of the samples were provided for subsequent protein analytics andanalytical characterization e.g. by SDS-PAGE, size exclusionchromatography, mass spectrometry and Endotoxin determination (see FIG.2).

SDS-PAGE

The NuPAGE® Pre-Cast gel system (Invitrogen) was used according to themanufacturer's instruction. In particular, 4-20% NuPAGE® Novex®TRIS-Glycine Pre-Cast gels and a Novex® TRIS-Glycine SDS running bufferwere used. (see e.g. FIG. 1). Reducing of samples was achieved by addingNuPAGE® sample reducing agent prior to running the gel.

Analytical Size Exclusion Chromatography

Size exclusion chromatography for the determination of the aggregationand oligomeric state of antibodies was performed by HPLC chromatography.Briefly, Protein A purified antibodies were applied to a Tosoh TSKgelG3000SW column in 300 mM NaCl, 50 mM KH2PO4/K2HPO4, pH 7.5 on an DionexHPLC system or to a Superdex 200 column (GE Healthcare) in 2×PBS on aDionex HPLC-System. The eluted protein was quantified by UV absorbanceand integration of peak areas. BioRad Gel Filtration Standard 151-1901served as a standard.

Mass Spectrometry

The total deglycosylated mass of antibodies was determined and confirmedvia electrospray ionization mass spectrometry (ESI-MS). Briefly, 100 μgpurified antibodies were deglycosylated with 50 mU N-Glycosidase F(PNGaseF, ProZyme) in 100 mM KH2PO4/K2HPO4, pH 7 at 37° C. for 12-24 hat a protein concentration of up to 2 mg/ml and subsequently desaltedvia HPLC on a Sephadex G25 column (GE Healthcare). The mass of therespective heavy and light chains was determined by ESI-MS afterdeglycosylation and reduction. In brief, 50 μg antibody in 115 μl wereincubated with 60 μl 1M TCEP and 50 μl 8 M Guanidinium-hydrochloridesubsequently desalted. The total mass and the mass of the reduced heavyand light chains was determined via ESI-MS on a Q-Star Elite MS systemequipped with a NanoMate source.

ANG-1 and ANG-2 Binding ELISA

The binding properties of antibodies directed against ANGPTs(Angiopoietin 1 or 2) were evaluated in an ELISA assay with full-lengthAngiopoietin-2-His protein (R&D Systems #623-AN/CF or in house producedmaterial) or Angiopoietin-1-His (R&D systems #923-AN). Therefore 96 wellplates (Falcon polystyrene clear enhanced microtiter plates or NuncMaxisorb) were coated with 100 μl 1 μg/mL recombinant humanAngiopoietin-1 or Angiopoietin-2 (carrier-free) in PBS (Sigma) for 2 hat room temperature or over night at 4° C. The wells were washed threetimes with 300 μl PBST (0.2% Tween 20) and blocked with 200 μl 2% BSA0.1% Tween 20 for 30 min at room temperature and subsequently washedthree times with 300 μl PBST. 100 μL/well of a dilution series (40pM-0.01 pM) of purified test antibody against <ANG-2> and as a referenceMab536 (Oliner, J., et al., Cancer Cell. November 6 (2004) 507-16, US2006/0122370) in PBS was added to the wells and incubated for 1 h on amicrotiterplate shaker at room temperature. The wells were washed threetimes with 300 μl PBST (0.2% Tween 20) and bound antibody was detectedwith 100 μL/well 0.1 μg/ml F(ab′)<hk>POD (Biozol Cat. No. 206005) in 2%BSA 0.1% Tween 20 as detection antibody for 1 h on a microtiterplateshaker at room temperature. Unbound detection antibody was washed awaythree times with 300 μL/well PBST and the bound detection antibody wasdetected by addition of 100 μL ABTS/well. Determination of absorbancewas performed on a Tecan Fluor Spectrometer at a measurement wavelengthof 405 nm (reference wavelength 492 nm).

ANG-2 Binding BIACORE

Binding of the antibodies to the antigen e.g. human ANG-2 wereinvestigated by surface plasmon resonance using a BIACORE T100instrument (GE Healthcare Biosciences AB, Uppsala, Sweden). Briefly, foraffinity measurements goat<hIgG-Fcgamma>polyclonal antibodies wereimmobilized on a CM4 chip via amine coupling for presentation of theantibodies against human ANG-2. Binding was measured in HBS buffer(HBS-P (10 mM HEPES, 150 mM NaCl, 0.05% Tween 20, ph 7.4), 25° C.Purified ANG-2-His (R&D systems or in house purified) was added invarious concentrations between 0.41 nM and 200 nM in solution.Association was measured by an ANG-2-injection of 3 minutes;dissociation was measured by washing the chip surface with HBS bufferfor 5 minutes and a KD value was estimated using a 1:1 Langmuir bindingmodel. Due to heterogenity of the ANG-2 preparation no 1:1 binding couldbe observed; KD values are thus only relative estimations. Negativecontrol data (e.g. buffer curves) were subtracted from sample curves forcorrection of system intrinsic baseline drift and for noise signalreduction. Biacore T100 Evaluation Software version 1.1.1 was used foranalysis of sensorgrams and for calculation of affinity data.Alternatively, Ang-2 could be captured with a capture level of 2000-1700RU via a PentaHisAntibody (PentaHis-Ab BSA-free, Qiagen No. 34660) thatwas immobilized on a CM5 chip via amine coupling (BSA-free) (see below).

Inhibition of huANG-2 Binding to Tie-2 (ELISA)

The interaction ELISA was performed on 384 well microtiter plates(MicroCoat, DE, Cat. No. 464718) at RT. After each incubation stepplates were washed 3 times with PBST. ELISA plates were coated with 0.5μg/ml Tie-2 protein (R&D Systems, UK, Cat. No. 313-TI) for at least 2hours (h). Thereafter the wells were blocked with PBS supplemented with0.2% Tween-20 and 2% BSA (Roche Diagnostics GmbH, DE) for 1 h. Dilutionsof purified antibodies in PBS were incubated together with 0.2 μg/mlhuAngiopoietin-2 (R&D Systems, UK, Cat. No. 623-AN) for 1 h at RT. Afterwashing a mixture of 0.5 μg/ml biotinylated anti-Angiopoietin-2 cloneBAM0981 (R&D Systems, UK) and 1:3000 diluted streptavidin HRP (RocheDiagnostics GmbH, DE, Cat. No. 11089153001) was added for 1 h.Thereafter the plates were washed 6 times with PBST. Plates weredeveloped with freshly prepared ABTS reagent (Roche Diagnostics GmbH,DE, buffer #204 530 001, tablets #11 112 422 001) for 30 minutes at RT.Absorbance was measured at 405 nm.

Inhibition of huANG-1 Binding to Tie-2 (ELISA)

The interaction ELISA was performed on 384 well microtiter plates(MaxiSorb Nunc#442768) at RT. After each incubation step plates werewashed 3 times with PBST. ELISA plates were coated with 0.5 μg/ml Tie-2protein (R&D Systems, UK, Cat. No. 313-TI or in house produced material)for at least 2 hours (h). Thereafter the wells were blocked with PBSsupplemented with 0.2% Tween-20 and 2% BSA (Roche Diagnostics GmbH, DE)for 1 h. Dilutions of purified antibodies in PBS were incubated togetherwith 0.2 μg/ml huAngiopoietin-1 (R&D Systems #923-AN/CF or in houseproduced material) for 1 h at RT. After washing a mixture of 0.5 μg/mlbiotinylated anti-Angiopoietin-1 clone (R&D Systems #BAF923) and 1:3000diluted streptavidin HRP (Roche Diagnostics GmbH, DE, Cat. No.11089153001) was added for 1 h. Thereafter the plates were washed 6times with PBST. Plates were developed with freshly prepared ABTSreagent (Roche Diagnostics GmbH, DE, buffer #204 530 001, tablets #11112 422 001) for 30 minutes at RT. Absorbance was measured at 405 nm.

Generation of HEK293-Tie2 Cell Line

In order to determine the interference of Angiopoietin-2 antibodies withANGPT2 stimulated Tie2 phosphorylation and binding of ANGPT2 to Tie2 oncells a recombinant HEK293-Tie cell line was generated. Briefly, apcDNA3 based plasmid (RB22-pcDNA3 Topo hTie2) coding for full-lengthhuman Tie2 (SEQ ID 61) under control of a CMV promoter and a Neomycinresistance marker was transfected using Fugene (Roche Applied Science)as transfection reagent into HEK293 cells (ATCC) and resistant cellswere selected in DMEM 10% FCS, 500 μg/ml G418. Individual clones wereisolated via a cloning cylinder, and subsequently analyzed for Tie2expression by FACS. Clone 22 was identified as clone with high andstable Tie2 expression even in the absence of G418 (HEK293-Tie2clone22). HEK293-Tie2 clone22 was subsequently used for cellular assays:ANGPT2 induced Tie2 phosphorylation and ANGPT2 cellular ligand bindingassay.

ANGPT2 Induced Tie2 Phosphorylation Assay

Inhibition of ANGPT2 induced Tie2 phosphorylation by ANGPT2 antibodieswas measured according to the following assay principle. HEK293-Tie2clone22 was stimulated with ANGPT2 for 5 minutes in the absence orpresence of ANGPT2 antibody and P-Tie2 was quantified by a sandwichELISA. Briefly, 2×105 HEK293-Tie2 clone 22 cells per well were grownover night on a Poly-D-Lysine coated 96 well-microtiter plate in 100 μlDMEM, 10% FCS, 500 μg/ml Geneticin. The next day a titration row ofANGPT2 antibodies was prepared in a microtiter plate (4-foldconcentrated, 75 μl final volume/well, duplicates) and mixed with 75 μlof an ANGPT2 (R&D systems #623-AN] dilution (3.2 μg/ml as 4-foldconcentrated solution). Antibodies and ANGPT2 were pre-incubated for 15min at room temperature. 100 μl of the mix were added to the HEK293-Tie2clone 22 cells (pre-incubated for 5 min with 1 mM NaV3O4, Sigma #S6508)and incubated for 5 min at 37° C. Subsequently, cells were washed with200 μl ice-cold PBS+1 mM NaV3O4 per well and lysed by addition of 120 μllysis buffer (20 mM Tris, pH 8.0, 137 mM NaCl, 1% NP-40, 10% glycerol, 2mM EDTA, 1 mM NaV3O4, 1 mM PMSF and 10 μg/ml Aprotinin) per well on ice.Cells were lysed for 30 min at 4° C. on a microtiter plate shaker and100 μA lysate were transferred directly into a p-Tie2 ELISA microtiterplate (R&D Systems, R&D #DY990) without previous centrifugation andwithout total protein determination. P-Tie2 amounts were quantifiedaccording to the manufacturer's instructions and IC50 values forinhibition were determined using XLfit4 analysis plug-in for Excel(Dose-response one site, model 205). IC50 values can be compared withinon experiment but might vary from experiment to experiment.

ANGPT1 Induced Tie2 Phosphorylation Assay

Inhibition of ANGPT1 induced Tie2 phosphorylation by ANGPT1 antibodieswas measured according to the following assay principle. HEK293-Tie2clone22 was stimulated with ANGPT1 for 5 minutes in the absence orpresence of ANGPT1 antibody and P-Tie2 was quantified by a sandwichELISA. Briefly, 2×105 HEK293-Tie2 clone 22 cells per well were grownover night on a Poly-D-Lysine coated 96 well-microtiter plate in 100 μlDMEM, 10% FCS, 500 μg/ml Geneticin. The next day a titration row ofANGPT1 antibodies was prepared in a microtiter plate (4-foldconcentrated, 75 μl final volume/well, duplicates) and mixed with 75 μlof an ANGPT1 (R&D systems #923-AN] dilution (0.8 μg/ml as 4-foldconcentrated solution). Antibodies and ANGPT1 were pre-incubated for 15min at room temperature. 100 μl of the mix were added to the HEK293-Tie2clone 22 cells (pre-incubated for 5 min with 1 mM NaV3O4, Sigma #S6508)and incubated for 5 min at 37° C. Subsequently, cells were washed with200 μl ice-cold PBS+1 mM NaV3O4 per well and lysed by addition of 120 μllysis buffer (20 mM Tris, pH 8.0, 137 mM NaCl, 1% NP-40, 10% glycerol, 2mM EDTA, 1 mM NaV3O4, 1 mM PMSF and 10 μg/ml Aprotinin) per well on ice.Cells were lysed for 30 min at 4° C. on a microtiter plate shaker and100 μl lysate were transferred directly into a p-Tie2 ELISA microtiterplate (R&D Systems, R&D #DY990) without previous centrifugation andwithout total protein determination. P-Tie2 amounts were quantifiedaccording to the manufacturer's instructions and IC50 values forinhibition were determined using XLfit4 analysis plug-in for Excel(Dose-response one site, model 205). IC50 values can be compared withinon experiment but might vary from experiment to experiment.

EXAMPLE 1 Expression & Purification of Monoclonal <ANG-2> AntibodiesAng2i-LC06, Ang2i-LC07 and Ang2k-LC08

Light and heavy chains of the corresponding antibodies Ang2i-LC06,Ang2i-LC07 and Ang2k-LC08 were constructed in expression vectors asdescribed above. The heavy chain and the kappa light was cloned in agenomic expression cassette, whereas the lambda light chain was clonedas cDNA with intron A (FIG. 1B). The plasmids were amplified in E. coli,purified, and subsequently transfected for transient expression ofrecombinant proteins in HEK293-F cells (utilizing Invitrogen's FreeStyle293 system). After 7 days, HEK 293-F cell supernatants were harvested,filtered and the antibodies were purified by protein A and sizeexclusion chromatography. Homogeneity of all antibodies was confirmed bySDS-PAGE under non reducing and reducing conditions and analytical sizeexclusion chromatography. Under reducing conditions (FIG. 1),polypeptide heavy chains of <ANG-2> antibodies showed upon SDS-PAGEapparent molecular sizes of ca. 50 kDa analogous to the calculatedmolecular weights, polypeptide light chains showed apparent molecularmasses of 25 kDa according to their predicted size. Mass spectrometryconfirmed the identity of the purified antibodies. Expression levels ofall constructs were analyzed by Protein A HPLC.

Size exclusion chromatography analysis of the purified. All antibodieswere prepared and analytically characterized analogously to theprocedure described. The SEC data of the corresponding antibodies weresummarized in the table below.

Anti- Theoretical Experimental SEC body mass mass (%) chain (Da) (Da)main peak <ANG-2>Ang- HC 50343 50325 (pyro-Glu) 99.7% 2i_LC07 LC 2273822720 (pyro-Glu) <ANG-2>Ang- HC 50343 50325 (pyro-Glu) 99.8% 2i_LC06 LC22620 22605 (pyro-Glu) <ANG-2>Ang- HC 49544 49527 (pyro-Glu) 99.8%2k_LC08 LC 22685 22667 (pyro-Glu)

EXAMPLE 2 ELISA Binding Assay to Human ANG-1 and to Human ANG-2

The binding of <ANG-2> antibodies Ang2i-LC06, Ang2i-LC07 and Ang2k-LC08to human ANG-1 and human ANG-2 was determined in an ANG-1 or ANG-2binding ELISA as described above. Briefly, the ELISA-type assay is basedon the immobilization of human wild-type Angiopoieti-1 or -2 in amicrotiter plate. Binding of an antibody directed against theimmobilized ANG-1 or ANG-2 is measured via an <human Fc> (anti-IgG)antibody with a POD conjugate. A dilution series of the <ANG-2>antibodyallows determining an EC50 concentration. As a reference the humananti-ANG-2 antibody <ANG-2> antibody Mab536 (Oliner et al., Cancer Cell.November 6 (2004) 507-16, US 2006/0122370) was used. The determined EC50concentrations are summarized in the table below.

hANG-1 binding hANG-2 binding Antibody EC50 EC50 <ANG-2>MAb536   2538ng/mL  133 ng/mL <ANG-2>Ang2i-LC06 >8000 ng/mL  84 ng/mL<ANG-2>Ang2i-LC07 >8000 ng/mL 3006 ng/mL <ANG-2>Ang2i-LC08   4044 ng/mL 105 ng/mL

All antibodies binds specifically to ANG-2. MAb536 and Ang2k-LC08 showalso specific binding towards ANG-1, whereas Ang2i-LC06 and Ang2i-LC07do not specifically bind to ANG-1 as they have an EC50-value of above8000 ng/ml (detection limit).

EXAMPLE 3 Binding to ANG-2 Via Biacore

The affinity for binding to human ANGPT2 was examined with a Biacoreassay as describes above. Briefly, is this assay a capturing antibody(anti-Fc) is immobilized to the surface of the Biacore chip, whichcaptures and presents the corresponding antibody (for exampleAng2i-LC06). The ligand (here ANGPT2) is captured from solution. Theaffinity for this interaction is determined with the assumption of a 1:1interaction. Details of this experiment can be found in the generalmethods section. The affinities determined for ANGPT2-binding (KD) aresummarized in the table below.

Average Experiment 1 Experiment 2 (from KD t_((1/2)) diss KD t_((1/2))diss 1 + 2) t_((1/2)) diss hAng-2 (pM) kd (1/s) (min) (pM) kd (1/s)(min) KD (pM) (min) Ang2i- 11 7.16E−05 161 21 1.14E−04 102 16 132 LC06Ang2k- 16 1.61E−04 72 27 2.28E−04 51 22 61 LC08 MAb536 29 1.44E−04 80 291.25E−04 92 29 86 The antibodies Ang2i-LC06 and Ang2k bind with highaffinity to ANGPT2.

EXAMPLE 4 Neutralization of ANGPT1/2-Tie2 Interaction (Human)

Blocking of human ANGPT1/2/human Tie2 interaction was shown by receptorinteraction ELISA. 384-well Maxisorp plates (Nunc) were coated with 0.5μg/ml human Tie2 (R&D Systems, UK, Cat. No. 313-TI or in house producedmaterial) for 2 h at room temperature and blocked with PBS supplementedwith 0.2% Tween-20 and 2% BSA (Roche Diagnostics GmbH, DE) for 1 h atroom temperature under shaking. In the meantime, Dilutions of purifiedantibodies in PBS were incubated together with 0.2 μg/ml huAngiopoietin-1/2 (R&D Systems #923-AN/CF, R&D Systems, UK, Cat. No. 623-AN or inhouse produced material) for 1 h at RT. After washing a mixture of 0.5μg/ml biotinylated anti-Angiopoietin-1/2 clone (R&D Systems #BAF923,BAM0981 R&D Systems, UK) and 1:3000 diluted streptavidin HRP (RocheDiagnostics GmbH, DE, Cat. No. 11089153001) was added for 1 h.Thereafter the plates were washed 6 times with PBST. Plates weredeveloped with freshly prepared ABTS reagent (Roche Diagnostics GmbH,DE, buffer #204 530 001, tablets #11 112 422 001) for 30 minutes at RT.Absorbance was measured at 405 nm.

The obtained inhibitory concentrations are summarized in the followingtable.

ANGPT1/Tie2 ANGPT2/Tie2 Antibody interaction ELISA interaction ELISAAng2i-LC06 >100 nM  0.1 nM Ang2k-LC08   11 nM 0.17 nM MAb536 n.d. 0.15nM

The table above shows different selectivity profiles for the twoantibodies Ang2i-LC06 and Ang2k-LC08. Ang2i-LC06 is ANGPT2 selective,whereas Ang2k-LC08 is ANGPT1/2 cross reactive in inhibition for ANGPT1/2Tie2 interaction.

EXAMPLE 5 Tie2 Phosphorylation

The ability of the identified ANGPT2 antibodies to interfere with ANGPT2and ANGPT1 mediated Tie2 phosphorylation was examined in the ANGPT2 andANGPT1 induced Tie2 phosphorylation assays as described above. Aschematic representation of the assay setup is depicted in FIG. 3.

Both antibodies Ang2i-LC06 and Ang2k-LC08 showed a dose-dependentinterference with ANGPT2 stimulated Tie2 phosphorylation as shown inFIG. 4 with comparable IC50 values. Ang2i-LC06 interfered with ANGPT2stimulated Tie2 phosphorylation with a IC50 value of approx. 508 ng/mland Ang2k-LC08 interfered with ANGPT2 stimulated Tie2 phosphorylationwith a IC50 value of approx. 499 ng/ml. In contrast, only Ang2k-LC08interfered with ANGPT1 stimulated Tie2 phosphorylation with a IC50 valueof approx. 391 ng/ml whereas Ang2i-LC06 did not interfere with ANGPT2stimulated Tie2 phosphorylation in the same tested concentration range(FIG. 5).

EXAMPLE 6 In Vivo Efficacy Effect of Anti ANGPT Antibodies on Colo205Xenograft Growth

In vivo efficacy of <ANGPT2> antibodies Ang2i-LC06 and Ang2k-LC08 incomparison to <ANGPT2> Mab536 in staged subcutaneous Colo205 xenograftmodel

The purified Ang2i-LC06 and Ang2k-LC08 antibodies were compared to theantibody Mab536 in the staged subcutaneous Colo205 xenograft model(Ang2_PZ_Colo205_006) in female Scid beige mice.

Antibodies: Mab536 was provided as frozen stock solution (c=4.5 mg/mL),Ang2i-LC06 and Ang2k-LC08 were provided as frozen stock solution (c=1mg/mL) in 20 mM Histidine, 140 mM NaCl, pH 6.0. Antibody solution wasdiluted appropriately in PBS from stock prior injections where requiredand PBS was applied as vehicle. The humanized IgG1 anti-IgE antibodyXolair (Omalizumab) served as positive control and was bought from apharmacy.

Cell lines and culture conditions: Colo205 human colorectal cancer cellswere originally obtained from ATCC and after expansion deposited in theRoche Penzberg internal cell bank. Tumor cell line was routinelycultured in RPMI 1640 medium (PAA, Laboratories, Austria) supplementedwith 10% fetal bovine serum (PAA Laboratories, Austria) and 2 mML-glutamine, at 37° C. in a water-saturated atmosphere at 5% CO2.Passage 3 was used for transplantation.

Animals: Female SCID beige mice (purchased from Charles River Germany)were maintained under specific-pathogen-free condition with daily cyclesof 12 h light/12 h darkness according to committed guidelines (GV-Solas;Felasa; TierschG). Experimental study protocol was reviewed and approvedby local government. After arrival animals were maintained in thequarantine part of the animal facility for one week to get accustomed tonew environment and for observation. Continuous health monitoring wascarried out on regular basis. Diet food (Provimi Kliba 3337) and water(acidified pH 2.5-3) were provided ad libitum. Age of mice at start ofthe study was about 12-14 weeks.

Monitoring: Animals were controlled daily for clinical symptoms anddetection of adverse effects. For monitoring throughout the experimentbody weight of animals was documented and tumor volume was measured bycaliper after staging.

Tumor cell injection: At day of injection Colo205 cells werecentrifuged, washed once and resuspended in PBS. After an additionalwashing with PBS cell concentration and cell size were determined usinga cell counter and analyzer system (Vi-CELL, Beckman Coulter). Forinjection of Colo205 cells, the final titer was adjusted to 5.0×10E7cells/ml, viability ca. 90%. Subsequently 100 μl of this suspensioncorresponding to 2.5*106 cells per animal was injected s.c. into theright flank of the mice.

Treatment of animals: Animal treatment started at day of randomisation,16 days after cell transplantation (study Ang2_PZ_Colo205_006) at a meantumor volume of 178 mm3.

Dose schedule of study Ang2_PZ_Colo205_006:

Cumu- Dose No of lative No of Com- (mg/ Route/Mode of treat- dose Groupanimals pound kg) administration ments (mg/kg) 1 10 Vehicle i.p. onceweekly 5 2 10 Xolair 10 i.p. once weekly 5 50 3 10 Ang2i- 10 i.p. onceweekly 5 50 LC06 5 10 Ang2k- 10 i.p. once weekly 5 50 LC08 6 10 MAB53610 i.p. once weekly 5 50

Tumor growth inhibition until Day 50 is shown in FIG. 6. The data showthat the ANGPT2 selective antibody Ang2i-LC06 was the most activeantibody (Tumor control ration (TCR) value 0.39). Ang2i-LC06 was moreefficacious in inhibiting tumor growth than antibody MAb536 (TCR value0.47) and the ANGPT2 selective, ANGPT1 cross-reactive antibodyAng2k-LC08 (TCR value 0.46).

Effect of Anti ANGPT Antibodies on KPL-4 Xenograft Growth

In vivo efficacy of <ANGPT2> antibodies Ang2i-LC06 and Ang2k-LC08 incomparison to <ANGPT2> Mab536 in staged orthotopic KPL-4 xenograftmodel. The purified Ang2i-LC06 and Ang2k-LC08 antibodies were comparedto the antibody Mab536 in the staged orthotopic KPL-4 xenograft model(Ang2_PZ_KPL-4_002) in female Scid beige mice.

Antibodies: Mab536 was provided as frozen stock solution (c=4.5 mg/mL),Ang2i-LC06 and Ang2k-LC08 were provided as frozen stock solution (c=1mg/mL) in 20 mM Histidine, 140 mM NaCl, pH 6.0. Antibody solution wasdiluted appropriately in PBS from stock prior injections where requiredand PBS was applied as vehicle.

Cell lines and culture conditions: KPL-4 human breast cancer cells wereoriginally established from the malignant pleural effusion of a breastcancer patient with an inflammatory skin metastasis. KPL-4 cells werekindly provided by Prof J. Kurebayashi (Kawasaki Medical School,Kurashiki, Japan). Tumor cells were routinely cultured in DMEM medium(PAN Biotech, Germany) supplemented with 10% fetal bovine serum (PANBiotech, Germany) and 2 mM L-glutamine (PAN Biotech, Germany) at 37° C.in a water-saturated atmosphere at 5% CO2. Culture passage was performedwith trypsin/EDTA 1× (PAN) splitting three times/week.

Animals: Female SCID beige mice (purchased from Charles River Germany)were maintained under specific-pathogen-free condition with daily cyclesof 12 h light/12 h darkness according to committed guidelines (GV-Solas;Felasa; TierschG). Experimental study protocol was reviewed and approvedby local government. After arrival animals were maintained in thequarantine part of the animal facility for one week to get accustomed tonew environment and for observation. Continuous health monitoring wascarried out on regular basis. Diet food (Provimi Kliba 3337) and water(acidified pH 2.5-3) were provided ad libitum. Age of mice at start ofthe study was about 12 weeks.

Monitoring: Animals were controlled daily for clinical symptoms anddetection of adverse effects. For monitoring throughout the experimentbody weight of animals was documented and tumor volume was measured bycaliper after staging.

Tumor cell injection: At the day of injection tumor cells were harvested(trypsin-EDTA) from culture flasks (Greiner TriFlask) and transferredinto 50 ml culture medium, washed once and resuspended in PBS. After anadditional washing step with PBS and filtration (cell strainer; Falcon™;100 μm) the final cell titer was adjusted to 1.5×108/ml. Tumor cellsuspension was carefully mixed with transfer pipette to avoid cellaggregation. Anesthesia was performed using a Stephens's inhalation unitfor small animals with preincubation chamber (plexiglas), individualmouse nose-mask (silicon) and not flammable or explosive anesthesiacompound Isoflurane (Pharmacia-Upjohn, Germany) in a closed circulationsystem. Two days before injection coat of the animals were shaved. Fori.m.f.p. injection cells were injected orthotopically at a volume of 20μl (3*106/animal) into the right penultimate inguinal mammary fat pad ofeach anesthetized mouse. For the orthotopic implantation, the cellsuspension was injected through the skin under the nipple using a usinga Hamilton microliter syringe and a 30 G×1/2″ needle.

Treatment of animals started at day of randomization with tumors rangingfrom 60-180 mm 3, 35 days after cell transplantation (studyAng2_PZ_KPL-4_002) at a mean tumor volume of ca. 90 mm3.

Dose schedule of study Ang2_PZ_KPL-4_002:

Cumu- Dose No of lative No of Com- (mg/ Route/Mode of treat- dose Groupanimals pound kg) administration ments (mg/kg) 1 10 Vehicle i.p. onceweekly 5 2 10 Xolair 10 i.p. once weekly 5 50 3 10 Ang2i- 10 i.p. onceweekly 5 50 LC06 5 10 Ang2k- 10 i.p. once weekly 5 50 LC08 6 10 MAB53610 i.p. once weekly 5 50

Tumor growth inhibition until day 64 is shown in FIG. 7. The data showthat the ANGPT2 selective antibody Ang2i-LC06 was the most activeantibody (TCR value 0.55) in the KPL-4 model. Ang2i-LC06 was moreefficacious in inhibiting tumor growth than antibody MAb536 (TCR value0.57) and the ANGPT2 selective, ANGPT1 cross-reactive antibodyAng2k-LC08 (TCR value 0.57).

EXAMPLE 7 Binding to ANG-1 Via Biacore

The affinity for binding to human ANG-1 was examined with a Biacoreassay: huAng-1 was immobilized on a CM5 biosensorchip usingamine-coupling chemistry. The protein was injected for 20 min in sodiumacetate pH 4.5 at 10 μg/ml at a flow rate of 5 μl/min. This resulted ina surface density of appr. 20000 RU. On the reference flow cell BSA wasimmobilized under the same conditions. The antibodies were diluted inHBS-P to 100 nM and injected for 3 min (association phase). Afterwashing with running buffer for 3 min (dissociation phase), the surfacewas regenerated by injecting 10 mM sodium hydroxide for 1 min at 5μl/min. Results are shown in FIG. 8: Ang2k_LC08 had a halftime ofcomplex dissociation of approximately 50 s, Ang2i_LC06 of appr. 5 s andAng2i_LC10 showed no binding to ANG-1.

EXAMPLE 8 Prevention of Metastasis/Secondary Tumors In Vivo in BearingPrimary Tumors

a) Prevention of Metastasis/Secondary in Mice Xenografted with PrimaryColo205 Tumors Cell Lines and Culture Conditions:

Colo205 human colorectal cancer cells were originally obtained from ATCCand after expansion deposited in the Roche Penzberg internal cell bank.Tumor cell line was routinely cultured in RPMI 1640 medium (PAA,Laboratories, Austria) supplemented with 10% fetal bovine serum (PAALaboratories, Austria) and 2 mM L-glutamine, at 37° C. in awater-saturated atmosphere at 5% CO₂. Passage 3 was used fortransplantation.

Animals:

Female SCID beige mice; age 4-5 weeks at arrival (purchased from CharlesRiver Germanyd) were maintained under specific-pathogen-free conditionwith daily cycles of 12 h light/12 h darkness according to committedguidelines (GV-Solas; Felasa; TierschG). Experimental study protocol wasreviewed and approved by local government. After arrival animals weremaintained in the quarantine part of the animal facility for one week toget accustomed to new environment and for observation. Continuous healthmonitoring was carried out on regular basis. Diet food (Provimi Kliba3337) and water (acidified pH 2.5-3) were provided ad libitum. Age ofmice at start of the study was about 10 weeks.

Tumor Cell Injection:

At the day of injection, Colo205 tumor cells were harvested(trypsin-EDTA) from culture flasks (Greiner) and transferred into 50 mlculture medium, washed once and resuspended in PBS. After an additionalwashing step with PBS and filtration (cell strainer; Falcon ø 100 μm)the final cell titer was adjusted to 2.5×10⁷/ml. Tumor cell suspensionwas carefully mixed with transfer pipette to avoid cell aggregation.After this, cell suspension was filled into a 1.0 ml tuberculin syringe(Braun Melsungen) using a wide needle (1.10×40 mm); for injection needlesize was changed (0.45×25 mm) and for every injection a new needle wasused. Anesthesia was performed using a Stephens inhalation unit forsmall animals with preincubation chamber (plexiglas), individual mousenose-mask (silicon) and not flammable or explosive anesthesia compoundIsoflurane (cp-pharma) in a closed circulation system. Two days beforeinjection coat of the animals were shaved and for cell injection skin ofanaesthetized animals was carefully lifted up with an anatomic forcepsand 100 μl cell suspension (=2.5×10⁶ cells) was injected subcutaneouslyin the right flank of the animals. Tumor growth of the primary tumorswas monitored (data not shown)

Monitoring of Secondary Tumors e.g. In the Lung by Quantification ofHuman Alu Sequences

At study termination (day 103) lungs were collected from animals of allgroups. Briefly, samples are transferred immediately into fluidnitrogen. In a further step total DNA was isolated from the samples withMagNA Pure LC Instrument according to manufacturer's instructions. HumanAlu specific primers were chosen for selective amplification of Alusequences by quantitative PCR (LightCycler instrument). (T. Schneideret. al., Clin. Exp. Metas. 2002; 19: 571-582).

Treatment of Animals

Treatment of animals with Avastin (10 mg/kg i.p. once weekly) wasstarted 14 days after cell transplantation (study Ang2_PZ_Colo205_008)at a mean tumor volume of 340 mm³. After 7 weeks mice were randomizedfor subsequent secondary treatment starting at day 51 with compoundslisted in table below. Secondary treatment starting at day 51 of StudyAng2_PZ_Colo205_008.

Route/ Mode of No of Cumulative No of Com- Dose adminis- treat- doseGroup animals pound (mg/kg) tration ments (mg/kg) 10 Avastin 10 mg/kgi.p. once 11 110 weekly 10 LC06 + 10 mg/kg i.p. once 6 60 Avastin weekly10 mg/kg i.p. once 11 110 weekly 10 LC06 10 mg/kg i.p. once 6 60 weeklyResults of Prevention of Metastasis/Secondary Tumors (in the Lung) areListed in the Table Below and Shown in FIG. 9A

TABLE 1 Quantification of human ALU DNA in the lungs of mice originallybearing primary Colo205 tumors, after treatment with differentantibodies Avastin + Ang2i- Avastin LC06 Ang2i_LC06 101 0.0264 2010.0042 301 0.0047 102 5.6740 202 0.0044 302 0.0055 103 0.0307 203 0.0065303 0.0050 104 0.0203 204 0.0081 304 0.0064 105 0.0215 205 0.0063 3050.0062 106 0.0338 206 0.0061 306 0.0066 107 0.0075 207 0.0053 307 0.0250108 0.0113 208 0.0506 308 0.0062 109 0.0087 209 0.0065 309 0.0067 1100.0587 210 0.0160 310 0.0064 mean 0.5893 0.0114 0.0079 median 0.02400.0064 0.0063Results show a clearly improved prevention of secondarytumors/metastasis by ANG2i-LC06 compared with Avastinb) Prevention of Metastasis/Secondary in Mice Xenografted with PrimaryKPL-4 Tumors Tumor Cell Line

The human breast cancer cell line KPL-4 (kindly provided by Prof. J.Kurebayashi) has been established from the malignant pleural effusion ofa breast cancer patient with an inflammatory skin metastasis. Tumorcells are routinely cultured in DMEM medium (PAN Biotech, Germany)supplemented with 10% fetal bovine serum (PAN Biotech, Germany) and 2 mML-glutamine (PAN Biotech, Germany) at 37° C. in a water-saturatedatmosphere at 5% CO₂. Culture passage is performed with trypsin/EDTA 1×(PAN) splitting three times/week.

Mice

After arrival, female SCID beige mice (age 10-12 weeks; body weight18-20 g) Charles River, Sulzfeld, Germany) were maintained in thequarantine part of the animal facility for one week to get themaccustomed to the new environment and for observation. Continuous healthmonitoring was carried out. The mice were kept under SPF-conditionsaccording to the international guidelines (GV-Solas; Felasa; TierschG)with daily cycles of 12 h light/12 h darkness. Diet food (Kliba Provimi3347) and water (filtered) were provided ad libitum. Experimental studyprotocol was reviewed and approved by the local government (Regierungvon Oberbayern; registration no. 211.2531.2-22/2003).

Tumor Cell Injection

At the day of injection tumor cells were harvested (trypsin-EDTA) fromculture flasks (Greiner TriFlask) and transferred into 50 ml culturemedium, washed once and resuspended in PBS. After an additional washingstep with PBS and filtration (cell strainer; Falcon Ø 100 μm) the finalcell titer was adjusted to 1.5×10⁸/ml. Tumor cell suspension wascarefully mixed with transfer pipette to avoid cell aggregation.Anesthesia is performed using a Stephens inhalation unit for smallanimals with preincubation chamber (plexiglas), individual mousenose-mask (silicon) and not flammable or explosive anesthesia compoundIsoflurane (Pharmacia-Upjohn, Germany) in a closed circulation system.Two days before injection coat of the animals were shaved. For i.m.f.p.injection cells were injected orthotopically at a volume of 20 μl intothe right penultimate inguinal mammary fat pad of each anesthetizedmouse. For the orthotopic implantation, the cell suspension was injectedthrough the skin under the nipple using a using a Hamilton microlitersyringe and a 30G×½″ needle. Tumor growth of the primary tumors wasmonitored (data not shown)

Monitoring of Secondary Tumors e.g. In the Lung by Quantification ofHuman Alu Sequences

At study termination (day 103) lungs were collected from animals of allgroups. Briefly, samples are transferred immediately into fluidnitrogen. In a further step total DNA was isolated from the samples withMagNA Pure LC Instrument according to manufacturer's instructions. HumanAlu specific primers were chosen for selective amplification of Alusequences by quantitative PCR (LightCycler instrument). (T. Schneideret. al., Clin. Exp. Metas. 2002; 19: 571-582)

Treatment of Animals

Treatment of animals was started 35 days after cell transplantation at amean tumor volume of 60-160 mm³. Compounds and dose schedule is listedin the table below.

Route/ Cumu- Mode of No of lative No of Dose adminis- treat- dose Groupanimals Compound (mg/kg) tration ments (mg/kg) 10 Vehicle i.p. twice 5weekly 10 Xolair 10 i.p. twice 5 50 weekly 10 Ang2i_LC06 10 i.p. once 440 weekly 10 Ang2i_LC07 10 i.p. once 4 40 weekly 10 Ang2k_LC08 10 i.p.once 4 40 weeklyResults of prevention of metastasis/secondary tumors (in the lung) arelisted in the table below and shown in FIG. 9B

TABLE 2 Quantification of human ALU DNA in the lungs of mice originallybearing primary KPL4 tumors, after treatment with different antibodiesVehicle Xolair Ang2i_LC06 Ang2i_LC07 Ang2i_LC08 101 0.0098 201 0.0157401 0.0273 501 0.0069 102 0.0090 202 0.0516 302 0.0076 402 0.0060 5020.0261 103 0.0119 203 0.0108 303 0.0413 403 0.0046 503 0.0067 104 0.0405204 0.0148 304 0.0042 404 0.0164 504 0.0044 205 0.0020 305 0.0041 4050.0040 505 0.0039 106 0.0381 206 0.0340 306 0.0093 406 0.0044 506 0.0051107 0.0281 207 0.0141 307 0.0038 407 0.0060 507 0.0037 208 0.0422 3080.0044 408 0.0174 508 0.0037 109 0.0121 209 0.0227 309 0.0036 409 0.0314509 0.0051 110 0.0143 210 0.0383 310 0.0094 410 0.0083 540 0.0200 median0.0132 0.0192 0.0044 0.0072 0.0051 mean 0.0205 0.0246 0.0098 0.01260.0086Results show a very efficient prevention of secondary tumors/metastasisby ANG2i-LC06, ANG2i-LC07, ANG2k-LC08.

EXAMPLE 9 Effects in the Treatment of Retinopathy

Methods

C57/B16 pups are cross fostered to CD1 nursing dams and are exposed to75% oxygen from P7 to P12 (PRO-OX 110 chamber oxygen controller,Biospherix Ltd, Redfield, N.Y.) which induces vessel obliteration andcessation of capillaries in the centre of the retina. The pups andnursing dams are placed in normal air leading to relative hypoxia andthe induction of neovascularisation. On P13, pups were anaesthetisedusing isofluorane (5% induction, 3% maintenance combined with 1.5%oxygen) and the eye was exposed and 1 μl intraocular injections using aNanofil syringe fitted with a 35 gauge needle (WPI, Sarasota, Fla.) intothe left eye was performed. On P17, both eyes were dissected, fixed in4% paraformaldehyde for 4 h at 4° C. and retinas were dissected. Retinaswere permeabilised in PBS containing 0.5% Triton X-100 and 1% bovineserum albumin, stained with 20 μg/ml biotinylated isolectin B4 (SigmaAldrich, Gillingham, UK) in PBS pH 6.8, 1% Triton-X100, 0.1 mM CaCl₂,0.1 mM MgCl₂, followed by 20 μg/ml ALEXA 488-streptavidin (MolecularProbes, Eugene, Oreg.) and flat mounted in Vectashield (VectorLaboratories, Burlingame, Calif.). Retinas were imaged using a Nikonepi-fluorescence microscope at 4× magnification. Quantification ofneovascular and ischaemic areas were performed in a blinded fashionusing Photoshop CS3 along with Image J (NiH) and expressed as percentageof total retinal area (=normal+ischaemic+neovascular).

Results

FIG. 10A show representative flat mounted retinas with the retinalvasculature visualised by isolectin staining. The centre ischemic areasinduce neovascularisation and re-growth of the retinal vessels byupregulation of angiogenic inducers. The neovascular front ishyperproliferative leading to tortuous vessels in an irregular vesselpattern. The most outer areas contain the normal unaffected vessels.Quantification of retinal flat mounts showed that inhibition of VEGFwith Avastin reduced retinal neovascularisation (see FIG. 10B,uninjected 36.7±1.8% to injected 22.4±3.0%) as expected. Inhibition ofAng2 using antibodies LC06 or LC08 also led to a reduction inneovascularisation (31.5±1.1% to 18.8±1.3% and 34.0±3.1% to 25.4±3.4%).Control injection of human Ig G had no effect on neovascularisation (seeFIG. 10B, 38.3±1.1% to 38.3±0.8%).

The invention claimed is:
 1. A method of inhibiting tumor growth in apatient comprising administering, to a patient in need thereof, anantibody which binds specifically to human angiopoietin-2 (ANG-2),wherein said antibody comprises: (A) a heavy chain variable domain whichcomprises the CDR3 region of SEQ ID NO: 1, the CDR2 region of SEQ ID NO:2 and the CDR1 region of SEQ ID NO: 3; and (B) a light chain variabledomain which comprises the CDR3 region of SEQ ID NO: 4, the CDR2 regionof SEQ ID NO: 5 and the CDR1 region of SEQ ID NO:
 6. 2. A methodaccording to claim 1 wherein said tumor is colorectal, breast, or lung.3. A method according to claim 1 wherein said heavy chain variabledomain has the sequence of SEQ ID NO: 7 and said light chain variabledomain has the sequence of SEQ ID NO:
 8. 4. A method for inhibitingmetastasis in a patient suffering from primary cancer comprisingadministering, to a patient in need of such treatment, an antibody whichbinds specifically to human angiopoietin-2 (ANG-2), wherein saidantibody comprises: (A) a heavy chain variable domain which comprisesthe CDR3 region of SEQ ID NO: 1, the CDR2 region of SEQ ID NO: 2 and theCDR1 region of SEQ ID NO: 3; and (B) a light chain variable domain whichcomprises the CDR3 region of SEQ ID NO: 4, the CDR2 region of SEQ ID NO:5 and the CDR1 region of SEQ ID NO:
 6. 5. A method according to claim 4wherein said heavy chain variable domain has the sequence of SEQ ID NO:7 and said light chain variable domain has the sequence of SEQ ID NO: 8.